Synthesis of Cyclic Analogs of the Binding Epitope of VCAM-1

Cyclization in solution:

a) The linear peptide H-Asn(Trt)-Ile-Asp(Ot-Bu)-Ser(t-Bu)-Pro-Leu-OH (72) is synthesized on 139 mg (100 µmol) Fmoc-Leu-Sasrin (loading 0.72 mmol/g) and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (72)): 95 mg, 65 µmol, 65 %.

HPLC (anal. method 1): tR=31.3 min, 68 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1012.58 [M+H]⁺, 1034.56 [M+Na]⁺, 1050.53 [M+K]⁺ Found: 1012.97 [M+H]⁺, 1034.63 [M+Na]⁺, 1050.78 [M+K]⁺

The linear peptide (72) is dissolved in 1 l DMF and cyclized following protocol 7, method A with different coupling reagents (Table 32). The cyclic protected peptide

(73) is purified by reverse phase preparative HPLC.

Table 32: Cyclization of H-Asn(Trt)-Ile-Asp(Ot-Bu)-Ser(t-Bu)-Pro-Leu-OH (72)

Linear peptide Yield

mg µmol

Coupling reagents

Time/

h mg µmol %

68 47 1.1 equiv PyAOP

3.0 equiv DIPEA 2x1 18 18 38

27 19 2.0 equiv DIC

1.0 equiv HOAt 24 - - -

27 19 1.1 equiv HATU

3.0 equiv DIPEA 0.5 5 5 26

HPLC (anal. method 1): tR=37.0 min.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 994.57 [M+H]⁺, 1016.55 [M+Na]⁺, 1032.52 [M+K]⁺ Found: 1016.86 [M+Na]⁺, 1032.87 [M+K]⁺

The combined quantities of cyclic protected peptide (73) are completely deprotected with the mixture TFA/H2O/TIS = 95 : 2.5 :2.5 for 3 h (protocol 13) and finally purified by preparative HPLC.

Yield (cyclic deprotected peptide (39)): 10 mg, 17 µmol, 74 %.

HPLC (anal. method 2): tR=17.3 min, 100 area %. No isomers detected.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 640.33 [M+H]⁺, 662.31 [M+Na]⁺, 678.29 [M+K]⁺ Found: 640.45 [M+H]⁺, 650.63 [M+Na]⁺, 678.44 [M+K]⁺

b) The linear peptide H-Asn(Trt)-Ile-Asp(Ot-Bu)-Ser(t-Bu)-Pro-Leu-OH (72) is synthesized on 2-ClTrt resin (295 mg, 200 µmol) previously loaded with Fmoc- -Leu-OH (loading 0.68 mmol/g) and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (72)): 213 mg, 173 µmol, 86 %.

HPLC (anal. method 1): tR=31.3 min, 82 area %.

MALDI-ToF MSmonoisotopic, m/z: see above.

The linear peptide (72) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (72) (213 mg, 173 µmol) in 20 ml DMF.

Second syringe: Solution of HATU (1.2 equiv, 200 µmol, 76 mg) in 20 ml DMF.

Addition rate for both syringes: 0.01 ml/min.

Flask: Solution of DIPEA (3.4 equiv, 585 µmol, 76 mg, 100 µl) and HOAt (1.2 equiv, 200 µmol, 27 mg) in 10 ml DMF.

The cyclic protected peptide (73) is purified by filtration through a C18 plug after evaporation of the solvent.

Yield (cyclic protected peptide (73)): 205 mg, 126 µmol, 73 %.

HPLC (anal. method 1): tR=37 min, 61 area %.

MALDI-ToF MSmonoisotopic, m/z: see above

The cyclic protected peptide (73) is completely deprotected with reagent K during 5 h (protocol 13) and finally purified by preparative HPLC.

Yield (cyclic deprotected peptide (39)): 30 mg, 46 µmol, 37 %.

HPLC (anal. method 2): tR=17.3 min, 100 area %. No isomers detected.

MALDI-ToF MSmonoisotopic, m/z: see above.

1H NMR (500 MHz, DMSO-d6), δ (ppm): two set of signals (ratio 3 : 1).

Major conformer: 0.77-0.86 (m, 12H, 3H^γ (Ile), 3H^δ (Ile), 6H^δ (Leu)), 1.18 (ddq,

2J=13.6 Hz, ³J=7.1 Hz, ³J=7.1 Hz, 1H, H^γ (Ile)), 1.41-1.55 (m, 4H, H^γ (Ile), 2H^β (Leu), H^γ(Leu)), 1.55-1.75 (m, 2H, H^β(Ile) H^β(Pro)), 1.82 (m, 1H, H^γ(Pro)), 1.93 (m, 1H, H^γ(Pro)), 2.23 (m, 1H, H^β(Pro)), 2.44 (dd, ²J=16.3 Hz, ³J=8.3 Hz 1H, H^β (Asx)), 2.67 (dd, ²J=16.6 Hz, ³J=2.0 Hz, 1H, H^β (Asx)), 2.78 (dd, ²J=16.6 Hz,

3J=7.8 Hz 1H, H^β(Asx)), 2.88 (dd, ²J=16.0 Hz, ³J=5.3 Hz 1H, H^β(Asx)), 3.54 (br, 1H, H^δ (Pro)), 3.65 (dd, ²J=15.1 Hz, ³J=3.1 Hz, 1H, H^β (Ser)), 3.79 (m, 1H, H^δ (Pro)), 3.84 (dd, ³J=11.9 Hz, ³J=5.0 Hz 1H, H^α(Ile)), 3.88 (dd, ²J=14.8 Hz, ³J=7.2 Hz 1H, H^β(Ser)), 4.12 (m, 1H, H^α(Leu)), 4.16 (dd, ³J=9.7 Hz, ³J=7.9 Hz, 1H, H^α (Pro)), 4.34 (ddd, ³J=7.5 Hz, ³J=6.6 Hz, ³J=1.9 Hz, 1H, H^α(Asx)), 4.54 (ddd, ³J=8.0 Hz, ³J=8.0 Hz, ³J=4.6 Hz, 1H, H^α(Asx)), 4.68 (m, 1H, H^α(Ser)), 7.40 (s, 1H, H^N (Asn)), 7.54 (d, ³J=6.3 Hz, 1H, H^N (Asx)), 7.61 (d, ³J=6.9 Hz, 1H, H^N (Ser)), 7.69 (d,

3J=8.8 Hz, 1H, H^N (Leu)), 7.81 (s, 1H, H^N (Asn)), 8.31 (br 1H, H^N (Asx)), 8.73 (d,

3J=5.0 Hz, 1H, H^N (Ile)), 12.3 (br, 1H, H^coo (Asp)).

Minor conformer 0.77-0.86 (m, 9H, 3H^γ (Ile), 3H^δ(Ile), 3H^δ(Leu)), 0.93 (d, ³J=6.3 Hz, 3H, H^δ(Leu)), 1.07 (ddq, ²J=13.6 Hz, ³J=7.2 Hz, ³J=2.1 Hz, 1H, H^γ(Ile)),1.31 (ddq, ²J=13.3 Hz, ³J=6.4 Hz, ³J=3.7 Hz, 1H, H^γ(Ile)), 1.41-1.55 (m, 2H, H^γ(Leu), H^γ (Pro)), 1.64 (m, 1H, H^β(Leu)), 1.76-1.87 (m, 3H, H^β(Leu), H^β(Pro), H^γ (Pro)), 1.88-1.99 (m, 2H, H^β(Ile), H^β(Pro)), 2.37 (dd, ²J=16.0 Hz, ³J=7.2 Hz, 1H, H^β(Asx)), 2.42 (dd, ²J=16.3 Hz, ³J=3.8 Hz, 1H, H^β(Asx)), 2.58-2.75 (m, 2H, H^β(Asx)), 3.39 (dt,

2J=11.1 Hz, ³J=5.7 Hz, 1H, H^δ(Pro)), 3.44-3.63 (br, 3H, H^δ(Pro), 2H^β(Ser)), 3.88 (m, 1H, H^α(Ile)), 4.06 (ddd, ³J=11.6 Hz, ³J=10.1 Hz, ³J=2.5 Hz, 1H, H^α(Leu)), 4.31 (m, 1H, H^α(Ser)), 4.41 (ddd, ³J=6.9 Hz, ³J=6.9 Hz, ³J=6.9 Hz, 1H, H^α(Asx)), 4.48 (ddd, ³J=6.9 Hz, ³J=6.9 Hz, ³J=6.9 Hz, 1H, H^α(Asx)), 4.67 (m, 1H, H^α(Pro)), 7.07 (s, 1H, H^N (Asn)), 7.12 (d, ³J=6.3 Hz, 1H, H^N (Asx)), 7.35 (d, ³J=8.2 Hz, 1H, H^N (Ile)), 7.47 (s, 1H, H^N (Asn)), 8.19 (d, ³J=3.8 Hz, 1H, H^N (Asx)), 8.53 (d, ³J=10.1 Hz, 1H, H^N (Leu)), 8.57 (br, 1H, H^N (Ser)), 12.3 (br, 1H, H^COO (Asp)).

c-(-Ile-Asp-Ser-ββββ-hPro-Leu-Asn-) (40)

C29H47N7O10 (653.74 g/mol).

Cyclization on resin:

Linear peptides H-Ser(t-Bu)-β-hPro-Leu-Asn(Trt)-Ile-Asp(Wang)-ODmb (74) and H-Ile-Asp(Ot-Bu)-Ser(t-Bu)-β-hPro-Leu-Asn(Rink amide)-ODmb are synthesized following the standard protocol (Table 30, P. 114) and cyclized on resin following protocol 6.

Table 33: On resin synthesis of c-(-Ile-Asp-Ser-ββββ-hPro-Leu-Asn-) (40)

Resin n/

mmol Coupling reagent Cleavage reagent

Time/

h Yield HPLC/

% Wang

(300 mg) 0.20 3.0 equiv HATU 6.0 equiv DIPEA

TFA/H2O/TIS 95 : 2.5 : 2.5 2

1 mg 2 µmol

1 % 100^a Rink

amide (170 mg)

0.10 3.0 equiv HATU

6.0 equiv DIPEA Reagent B 1 0 0

a The same HPLC analytical method and the retention time as for the cyclization in solution.

After deprotection and cleavage from the resin with different cleavage reagents (Table 33) the cyclic peptide (40) is purified by reverse phase preparative HPLC:

Cyclization in solution:

The linear peptide H-Asn(Trt)-Ile-Asp(Ot-Bu)-Ser(t-Bu)-β-hPro-Leu-OH (75) is synthesized on 280 mg (0.20 mmol) Fmoc–Leu-Sasrin (loading 0.72 mmol/g) and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (75)): 134 mg, 0.12 mmol, 60 %.

HPLC (anal. method 1): tR=29.8 min, 90 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1026.59 [M+H]⁺, 1048.57 [M+Na]⁺, 1064.55 [M+K]⁺ Found: 1026.94 [M+H]⁺, 1048.87 [M+Na]⁺, 1065.91 [M+K]⁺ The linear peptide (75) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (75) (180 mg, 0.12 mmol) in 10 ml DMF.

Second syringe: Solution of HATU (3.0 equiv, 0.36 mmol, 137 mg) in 10 ml DMF.

Addition rate for both syringes: 0.04 ml/min.

Flask: Solution of DIPEA (6.0 equiv, 0.72 mmol, 93 mg, 123 µl) and HATU (0.1 equiv, 12 µmol, 5 mg) in 10 ml DMF.

The cyclic protected peptide (76) is purified by reverse phase preparative HPLC after evaporation of the solvent.

Yield (cyclic protected peptide (76)): 72 mg, 72 µmol, 60 %.

HPLC (anal. method 1): tR=35.8 min, 100 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1008.58 [M+H]⁺, 1030.56 [M+Na]⁺, 1046.54 [M+K]⁺ Found: 1030.42 [M+Na]⁺, 1046.72 [M+K]⁺

The cyclic protected peptide (76) is completely deprotected with reagent K during 4 h (protocol 13) and finally purified by preparative HPLC.

Yield (cyclic deprotected peptide (40)): 47 mg, 71 µmol, 99 %.

HPLC (anal. method 2): tR=16.7 min, 100 area %. No isomers detected.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 654.35 [M+H]⁺, 676.33 [M+Na]⁺, 692.30 [M+K]⁺ Found: 654.52 [M+H]⁺, 676.56 [M+Na]⁺, 692.50 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two set of signals (ratio 7 : 1).

0.80 (d, ³J=6.3 Hz, 3H, H^δ(Leu)), 0.82 (dd, ³J=7.2 Hz, ³J=7.2 Hz, 3H, H^δ(Ile)), 0.84 (d, ³J=6.3 Hz, 3H, H^γ(Ile)), 0.86 (d, ³J=6.3 Hz, 3H, H^δ(Leu)), 1.19 (ddq, ²J=15.5 Hz, ³J=7.1 Hz, ³J=7.2 Hz, 1H, H^γ(Ile)), 1.37-1.48 (m, 2H, H^γ (Ile), H^β(Leu)), 1.54-1.64 (m, 2H, H^γ (Leu), H^γ (β-hPro)), 1.69-1.87 (m, 4H, H^β (Ile), H^β (Leu), 2H^δ (β-hPro)), 2.06 (dddd, ²J=11.9 Hz, ³J=6.4 Hz, ³J=6.4 Hz, ³J=5.4 Hz, 1H, H^γ (β-hPro)), 2.16 (dd, ²J=14.4 Hz, ³J=1.9 Hz, 1H, H^α(β-hPro)), 2.50-2.56 (m, 2H, H^β

(Asx), H^α(β-hPro)), 2.67-2.73 (m, 2H, 2H^β(Asx)), 2.83 (dd, ²J=15.4 Hz, ³J=4.1 Hz, 1H, H^β(Asx)), 3.43 (dd, ²J=10.4 Hz, ³J=8.5 Hz, 1H, H^β(Ser)), 3.51 (dd, ²J=10.4 Hz,

3J=4.7 Hz, 1H, H^β(Ser)), 3.52 (m, 1H, H^ε(β-hPro)), 3.59 (ddd, ²J=10.1 Hz, ³J=7.9 Hz, ³J=7.9 Hz, 1H, H^ε(β-hPro)), 3.69 (dd, ³J=5.0 Hz, ³J=3.6 Hz, 1H, H^α(Ile)), 4.11 (dddd, ³J=7.5 Hz, ³J=7.5 Hz, ³J=6.3 Hz, ³J=1.9 Hz, 1H, H^β(β-hPro)), 4.25 (ddd,

3J=8.6 Hz, ³J=6.4 Hz, ³J=3.0 Hz, 1H, H^α(Leu)), 4.53-4.56 (m, 2H, H^α (Asx), H^α (Ser)), 4.72 (ddd, ³J=8.2 Hz, ³J=4.7 Hz, ³J=4.7 Hz, 1H, H^α(Asx)), 7.1 (d, ³J=7.5 Hz, 1H, H^N (Ser)), 7.19 (s, 1H, H^N (Asx)), 7.62 (s, 1H, H^N (Asx)), 7.79 (d, ³J=8.8 Hz, 1H, H^N (Leu)), 8.14 (d, ³J=9.4 Hz, 1H, H^N (Asx)), 8.17 (d, ³J=3.2 Hz, 1H, H^N (Ile)), 8.48 (d, ³J=8.2 Hz, 1H, H^N (Asx)).

c-(-Ile-Asp-ββββ-hSer-Pro-Leu-ββββ-hAsn-) (41)

C30H49N7O10 (667.77 g/mol).

Cyclization on resin:

The linear peptide H-β-hSer-Pro-Leu-β-hAsn(Trt)-Ile-Asp(Wang)-ODmb (77) is synthesized on Wang resin (170 mg, 90 µmol), and cyclized following protocol 6 (HATU as coupling reagent). After complete deprotection and cleavage from the resin with the cleavage cocktail, TFA/DCM/TIS = 48 : 48 : 4 during 2 h, (protocol 10), the cyclic peptide (41) is purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (41)): 3 mg, 4 µmol, 4 %.

HPLC (anal. method 2): tR=14.5 min, 85 area %.

Cyclization in solution:

The linear peptide H-β-hAsn(Trt)-Ile-Asp(Ot-Bu)-β-hSer(t-Bu)-Pro-Leu-OH (78) is synthesized on 280 mg (0.20 mmol) Fmoc–Leu-Sasrin (loading 0.72 mmol/g) and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (78)): 180 mg, 0.14 mmol, 70 %.

HPLC (anal. method 1): tR=29.2 min, 80 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1040.61 [M+H]⁺, 1062.59 [M+Na]⁺, 1078.56 [M+K]⁺ Found: 1040.79 [M+H]⁺, 1062.73 [M+Na]⁺, 1078.75 [M+K]⁺ The linear peptide (78) is cyclized following protocol 7, method B.

First syringe: Solution of linear peptide (78) (180 mg, 0.14 mmol) in 15 ml DMF.

Second syringe: Solution of HATU (1.1 equiv, 0.15 mmol, 59 mg) in 15 ml DMF.

Addition rate for both syringes: 0.02 ml/min.

Flask: Solution of DIPEA (3.0 equiv, 0.42 mmol, 54 mg, 72 µl) and HATU (0.5 equiv, 70 µmol, 27 mg) in 20 ml DMF.

The cyclic protected peptide (79) is purified by reverse phase preparative HPLC after evaporation of the solvent.

Yield (cyclic protected peptide (79)): 78 mg, 76 µmol, 54 %.

HPLC (anal. method 1): tR=34.5 min, 100 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1022.60 [M+H]⁺, 1044.58 [M+Na]⁺, 1060.55 [M+K]⁺ Found: 1044.67 [M+Na]⁺, 1060.88 [M+K]⁺

The cyclic protected peptide (79) is completely deprotected with the cleavage mixture TFA/H2O/TIS = 95 : 2.5 : 2.5 during 1 h (protocol 13) and finally purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (41)): 33 mg, 49 µmol, 65 %.

HPLC (anal. method 2): tR=14.5 min, 100 area %. No isomers detected.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 668.36 [M+H]⁺, 690.34 [M+Na]⁺, 706.32 [M+K]⁺ Found: 668.64 [M+H]⁺, 690.62 [M+Na]⁺, 706.58 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two set of signals (ratio 6 : 1)

0.76 (d, ²J=6.9 Hz, 3H, H^δ(Leu)), 0.82 (dd, ³J=7.5 Hz, ³J=7.5 Hz, 3H, H^δ(Ile)), 0.86 (d, ³J=6.9 Hz, 3H, H^γ(Ile)), 0.87 (d, ³J=6.3 Hz, 3H, H^δ(Leu)), 1.23 (m, 1H, H^γ(Ile)), 1.33-1.63 (m, 5H, H^γ(Ile), 2H^β(Leu), H^γ(Leu), H^γ(Pro)), 1.73 (m, 1H, H^β(Ile)), 1.83 (m, 1H, H^γ(Pro)), 2.00-2.11 (m, 2H, H^β(Pro)), 2.22-2.31 (m, 2H, H^α (β-hAsn), H^γ (β-hAsn)), 2.33-2.40 (m, 2H, H^γ(β-hAsn), H^β(Asp)), 2.57 (dd, ²J=16.9 Hz, ³J=10.7 Hz, 1H, H^α(β-hSer)), 2.63 (dd, ²J=15.7 Hz, ³J=5.7 Hz, 1H, H^α(β-hAsn)), 2.75 (dd,

2J =16.6 Hz, ³J =2.8 Hz, 1H, H^α(β-hSer)), 2.86 (dd, ²J=16.0 Hz, ³J=11.6 Hz, 1H, H^β(Asp)), 3.30 (ddd, ²J=11.3 Hz, ³J=9.7 Hz, ³J=9.4 Hz, 1H, H^δ (Pro)), 3.34 (dd,

2J=11 Hz, ³J=3.4 Hz, 1H, H^γ(β-hSer)), 3.42 (ddd, ²J=11.2 Hz, ³J= 11.2 Hz, ³J= 7.1 Hz, 1H, H^δ (Pro)), 3.67 (dd, ²J=10.7 Hz, ³J=3.8 Hz, 1H, H^γ (β-hSer)), 3.74 (dd,

3J=3.8 Hz, ³J=3.8 Hz, 1H, H^α(Ile)), 4.04 (m, 1H, H^α(Asp)), 4.12 (ddd, ²J=4.2 Hz,

2J=7.1 Hz, ²J= 11.2 Hz, 1H, H^α(Leu)), 4.25 (m, 1H, H^β(β-hAsn)), 4.40-4.49 (m, 2H, H^β(β-hSer), H^α (Pro)), 7.19 (s, 1H, H^N (β-hAsn)), 7.45 (d, ³J=7.5 Hz, 1H, H^N (Asp)), 7.63 (d, ³J=8.8 Hz, 1H, H^N (β-hAsn)), 7.66 (m, 2H, H^N (β-hAsn), H^N (Leu)), 8.34 (d,

3J=3.8 Hz, 1H, H^N (Ile)),8.40 (d, J=7.5 Hz, 1H, H^N (β-hSer)).

c-(-Val-Asp-Ser-Pro-Leu-Asn-) (42)

C27H43N7O10 (625.69 g/mol).

Cyclization in solution:

The linear peptide H-Asn(Trt)-Val-Asp(Ot-Bu)-Ser(t-Bu)-Pro-Leu-OH (80) is synthesized on 2-ClTrt resin (300 mg, 0.20 mmol) previously loaded with Fmoc- -Leu-OH (loading 0.67 mmol/g), cleaved with 1 % TFA in DCM (protocol 9), and purified by preparative HPLC.

Yield (linear protected peptide (80)): 90 mg, 72 µmol, 36 %.

HPLC (anal. method 1): tR=29.1 min, 80 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 998.56 [M+H]⁺, 1020.54 [M+Na]⁺, 1036.52 [M+K]⁺ Found: 998.66 [M+H]⁺, 1020.56 [M+Na]⁺, 1036.58 [M+K]⁺ The linear peptide (80) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (80) (90 mg, 72 µmol) in 10 ml DMSO.

Second syringe: Solution of HATU (1.0 equiv, 72 µmol, 27mg) in 10 ml DMSO.

Addition rate for both syringes: 0.02 ml/min.

Flask: Solution of DIPEA (3.0 equiv, 216 µmol, 28 mg, 37 µl) and HOAt (0.1 equiv, 7 µmol 10 mg) in 10 ml DMSO.

After cyclization, the cyclic protected peptide (81) is precipitated with 200 ml H2O and filtered through a C18 plug. The peptide is washed with water (3x) and Et2O (3x) and eluted from the plug with DCM.

Yield (cyclic protected peptide (81)): 75 mg, 61 µmol, 85 %.

HPLC (anal. method 1): tR=34.5 min, 34.8 min, 80 area % sum of epimers.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 980.55 [M+H]⁺, 1002.53 [M+Na]⁺, 1018.51 [M+K]⁺ Found: 1002.30 [M+Na]⁺, 1018.59 [M+K]⁺

The cyclic protected peptide (81) is completely deprotected with reagent K during 5 h (protocol 13) and finally purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (42)): 29 mg, 47 µmol, 77 %.

HPLC (anal. method 2): tR=16.0 min, 16.6 min, 100 area %, epimers ratio 65:35.

For the biological tests and the NMR analysis the epimers were separated by reverse phase preparative HPLC (preparative method 3).

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 626.31 [M+H]⁺, 648.30 [M+Na]⁺, 664.27 [M+K]⁺ Found: 626.56 [M+H]⁺, 648.54 [M+Na]⁺, 664.52 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): 0.79 (d, ³J=6.3 Hz, 3H, H^δ (Leu)), 0.85-0.90 (m, 6H, 3H^δ (Leu), 3H^γ(Val)), 0.92 (d, ³J=6.9 Hz, 3H, H^γ(Val)), 1.49 (m, 1H,

H^β (Leu)), 1.60-1.69 (m, 2H, H^β (Leu), H^γ (Leu)), 1.71-1.86 (m, 2H, H^β (Pro), H^γ (Pro)), 1.89-2.07 (m, 3H, H^β(Pro), H^γ(Pro), H^β(Val)), 2.30 (dd, ²J=16.0 Hz, ³J=2.8 Hz, 1H, H^β (Asx)), 2.45 (dd, ²J=15.4 Hz, ³J=8.8 Hz, 1H, H^β (Asx)), 2.76 (dd,

2J=15.4 Hz, ³J=5.3 Hz, 1H, H^β (Asx)), 2.82 (dd, ²J=15.7 Hz, ³J=8.8 Hz, 1H, H^β (Asx)), 3.48 (ddd, ²J=9.7 Hz, ³J=9.7 Hz, ³J=6.3 Hz, 1H, H^δ(Pro)), 3.53 (dd, ²J=11.3 Hz, ³J=4.4 Hz, 1H, H^β(Ser)), 3.61 (dd, ²J=11.3 Hz, ³J=4.4 Hz, 1H, H^β(Ser)), 3.73 (ddd, ²J=10.1 Hz, ³J=7.2 Hz, ³J=2.5 Hz, 1H, H^δ(Pro)), 3.77 (dd, ³J=7.5 Hz, ³J=5.7 Hz, 1H, H^α(Val)), 4.04 (ddd, ³J=10.9 Hz, ³J=8.2 Hz, ³J=2.5 Hz, 1H, H^α(Leu)), 4.18 (dd, ³J=8.5 Hz, ³J=7.2 Hz, 1H, H^α(Pro)), 4.45 (ddd, ³J=8.6 Hz, ³J=8.6 Hz, ³J=2.3 Hz, 1H, H^α(Asx)), 4.52 (ddd, ³J=6.6 Hz, ³J=4.7 Hz, ³J=4.7 Hz, 1H, H^α(Ser)), 4.65 (ddd, ³J=8.8 Hz, ³J=8.8 Hz, ³J=5.0 Hz, 1H, H^α(Asx)), 7.24 (s, 1H, H^N (Asn)), 7.35 (d, ³J=6.9 Hz, 1H, H^N (Ser)), 7.65 (s, 1H, H^N (Asn)), 8.10 (d, ³J=8.8 Hz, 1H, H^N (Asx)), 8.43 (d, ³J=8.2 Hz, 1H, H^N (Asx)), 8.74 (d, ³J=8.2 Hz, 1H, H^N (Leu)), 9.07 (d, ³J=5.7 Hz, 1H, H^N (Val)), 12.32 (br, 1H, H^COO (Asp)).

c-(-D-Val-Asp-Ser-Pro-Leu-Asn-) (43)

C27H43N7O10 (625.69 g/mol).

Cyclization on resin:

The linear peptide H-Ser(t-Bu)-Pro-Leu-Asn(Trt)-D-Val-Asp(Wang)-ODmb (82) is synthesized on Wang resin (400 mg, 0.20 mmol) and cyclized following protocol 6 (3.0 equiv HATU and 6.0 equiv DIPEA as the cleavage reagents). After complete deprotection and cleavage from the resin with the cleavage cocktail TFA/DCM/TIS = 48 : 48 : 4 during 2 h, (protocol 10) the cyclic peptide (43) is purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (43): 9 mg, 14 µmol, 7 %.

HPLC (anal. method 2): tR=16.3 min, 100 area %.

Cyclization in solution:

The linear peptide H-Leu-Asn(Trt)-D-Val-Asp(Ot-Bu)-Ser(t-Bu)-Pro-OH (83) is synthesized on 2-ClTrt resin (260 mg 0.20 mmol) previously loaded with Fmoc-Pro-OH (loading 0.76 mmol/g), and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (83)): 133 mg, 99 µmol, 50 %.

HPLC (anal. method 1): tR=31.5 min, 74 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 998.56 [M+H]⁺, 1020.54 [M+Na]⁺, 1036.52 [M+K]⁺ Found: 998.74 [M+H]⁺, 1020.74 [M+Na]⁺, 1036.77 [M+K]⁺ The linear peptide (83) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (83) (133 mg, 99 µmol) in 15 ml DMF.

Second syringe: Solution of HATU (2.0 equiv, 200 µmol, 76 mg) in 15 ml DMF.

Addition rate for both syringes: 0.01 ml/min.

Flask: Solution of DIPEA (6.0 equiv, 600 µmol, 77 mg, 100 µl) and HOAt (3.0 equiv, 200 µmol, 27 mg) in 20 ml DMF.

The cyclic protected peptide (84) is purified by filtration through a C18 plug after removing the DMF.

Yield (cyclic protected peptide (84)): 59 mg, 60 µmol, 61 %.

HPLC (anal. method 1): tR=35.7 min, 77 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 980.55 [M+H]⁺, 1002.53 [M+Na]⁺, 1018.51 [M+K]⁺ Found: 1002.45 [M+Na]⁺, 1018.53 [M+K]⁺

The cyclic protected peptide is completely deprotected with reagent K during 5 h (protocol 13) and finally purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (43)): 32 mg, 51 µmol, 85 %.

HPLC (anal. method 2): tR=16.3 min, 100 area %. No isomers detected.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 626.31 [M+H]⁺, 648.30 [M+Na]⁺, 664.27 [M+K]⁺ Found: 626.59 [M+H]⁺, 648.45 [M+Na]⁺, 664.48 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two sets of signals (ratio 6 : 1).

0.78 (d, ³J=6.3 Hz, 3H, H^γ (D-Val)), 0.79 (d, ³J=6.3 Hz, 3H, H^δ (Leu)), 0.86 (d,

3J=6.3 Hz, 3H, H^δ (Leu)), 0.91 (d, ³J=6.3 Hz, 3H, H^γ (D-Val)), 1.44-1.55 (m, 3H, 2H^β (Leu), H^γ(Leu)), 1.64 (m, 1H, H^β (Pro)), 1.80-1.90 (m, 2H, H^γ(Pro), H^β(D-Val)), 1.94 (m, 1H, H^γ(Pro)), 2.18 (m, 1H, H^β(Pro)), 2.23 (dd, ²J=15.1 Hz, ³J=3.8 Hz, 1H, H^β(Asx)), 2.48 (br, 1H, H^β(Asx)), 2.74 (dd, ²J=15.1 Hz, ³J=10.0 Hz, 1H, H^β(Asx)), 2.78 (dd, ²J=16.6 Hz, ³J=4.1 Hz, 1H, H^β(Asx)), 3.55 (dd, ³J=9.7 Hz, ³J=5.3 Hz, 1H, H^α(D-Val)), 3.63-3.71 (br, 2H, H^δ (Pro)), 3.83 (dd, ²J=11.9 Hz, ³J=3.8 Hz, 1H, H^β (Ser)), 3.93 (dd, ²J=11.6 Hz, ³J=6.6 Hz, 1H, H^β(Ser)), 4.01 (dd, ³J=8.5 Hz, ³J=7.9 Hz, 1H, H^α(Pro)), 4.08 (ddd, ³J=8.6 Hz, ³J=8.6 Hz, ³J=5.8 Hz, 1H, H^α(Leu)), 4.49 (ddd, ³J=9.6 Hz, ³J=5.5 Hz, ³J=4.2 Hz, 1H, H^α (Asx)), 4.57 (ddd, ³J=10.2 Hz,

3J=8.7 Hz, ³J=3.9 Hz, 1H, H^α(Asx)), 4.72 (ddd, ³J=9.6 Hz, ³J=6.1 Hz, ³J=3.6 Hz, 1H, H^α(Ser)), 6.83 (s, 1H, H^N (Asn)), 6.97 (d, ³J=5.7 Hz, 1H, H^N (Asx)), 7.22 (s, 1H, H^N (Asn)), 7.44 (d, ³J=8.8 Hz, 1H, H^N (Leu)), 7.95 (d, ³J=9.4 Hz, 1H, H^N (Ser)), 8.7

(d, ³J=8.2 Hz, 1H, H^N (Asx)), 8.7 (d, ³J=5.7 Hz, 1H, H^N (D-Val)), 12.30 (br, 1H, H^COO (Asp)).

c-(-Val-Asp-Ser-ββββ-hPro-Leu-Asn-) (44)

C28H45N7O10 (639.72 g/mol).

Cyclization on resin:

The linear peptide H-Ser(t-Bu)-β-hPro-Leu-Asn(Trt)-Val-Asp(Wang)-ODmb (85) is synthesized and cyclized (protocol 6) on Wang resin. After complete deprotection and cleavage from the resin with different cleavage cocktails (Table 34, protocol 10) the cyclic peptide (44) is purified by reverse phase preparative reverse phase HPLC:

Table 34: On resin cyclization of H-Ser(t-Bu)-ββββ-hPro-Leu-Asn(Trt)-Val-Asp(Wang)-ODmb (85)

Resin n/

mmol Coupling reagent t/

min

Cleavage reagent

t/

h Yield

HPLC /

% Wang

280 mg 0.20 3.0 equiv HATU

6.0 equiv DIPEA 45 Reagent B 5

8 mg 6 µmol

3 %

50

Wang

400 mg 0.20 3.0 equiv HATU 6.0 equiv DIPEA 45

TFA 48 DCM 48

TIS 4

2

10 mg 16 µmol

8 %

100

HPLC (anal. method 2): tR=17.0 min.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 640.33 [M+H]⁺, 662.31 [M+Na]⁺, 678.29 [M+K]⁺ Found: 640.42 [M+H]⁺, 662.55 [M+Na]⁺, 678.45 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): two sets of signals (ratio 8 : 1).

0.79 (d, ³J=6.3 Hz, 3H, H^δ(Leu)), 0.86 (d, ³J=6.3 Hz, 3H, H^δ(Leu)), 0.88 (d, ³J=6.9 Hz, 3H, H^γ(Val)), 0.90 (d, ³J=6.9 Hz, 3H, H^γ(Val)), 1.44 (ddd, ²J=13.5 Hz, ³J=11.9 Hz, ³J=3.5 Hz, 1H, H^β(Leu)), 1.53-1.62 (m, 2H, H^γ(Leu), H^γ(β-hPro)), 1.69-1.85 (m, 3H, H^β(Leu), 2H^δ(β-hPro)), 1.98-2.09 (m, 2H, H^γ(β-hPro), H^β(Val)), 2.16 (dd,

2J=14.4 Hz, ³J=1.9 Hz, 1H H^α (β-hPro)), 2.49 (br, 1H, H^α (β-hPro)), 2.52 (dd,

2J=16.3 Hz, ³J=10.0 Hz, 1H, H^β (Asx)), 2.67 (dd, ²J=15.7 Hz, ³J=3.8 Hz, 1H, H^β (Asx)), 2.69 (dd, ²J=15.1 Hz, ³J=5.7 Hz, 1H, H^β (Asx)), 2.85 (dd, ²J=15.4 Hz,

3J=4.1 Hz, 1H, H^β(Asx)), 3.42 (m, 1H, H^β(Ser)), 3.47-3.54 (m, 2H, H^ε (β-hPro), H^β (Ser)), 3.57 (m, 1H, H^ε (β-hPro)), 3.61 (dd, ³J=4.1 Hz, ³J=4.7 Hz, 1H, H^α(Val)), 4.10 (m, 1H, H^β (β-hPro)), 4.24 (ddd, ³J=11.9 Hz, ³J=8.8 Hz, ³J=3.1 Hz, 1H, H^α (Leu)), 4.52-4.61 (m, 2H, H^α (Asx), H^α (Ser)), 4.73 (ddd, ³J=7.9 Hz, ³J=4.7 Hz,

3J=4.7 Hz, 1H, H^α(Asx)), 4.87 (br, 1H, H^O (Ser)), 7.11 (d, ³J=7.5 Hz, 1H, H^N (Ser)), 7.24 (s, 1H, H^N (Asn)), 7.67 (s, 1H, H^N (Asn)), 7.82 (d, ³J=8.8 Hz, 1H, H^N (Leu)), 8.16 (d, ³J=3.8 Hz, 1H, H^N (Val)), 8.17 (d, ³J=10.1 Hz, 1H, H^N (Asx)), 8.5 (d, ³J=8.2 Hz, 1H, H^N (Asx)), 12.33 (br, 1H, H^COO (Asp)).

c-(-D-Val-Asp-Ser-ββββ-hPro-Leu-Asn-) (45)

C28H45N7O10 (639.72 g/mol).

Cyclization on resin:

The linear peptide H-Ser(t-Bu)-β-hPro-Leu-Asn(Trt)-D-Val-Asp(Wang)-ODmb (86)

is synthesized and cyclized following protocol 6 (3.0 equiv HATU and 6.0 equiv DIPEA as the cleavage reagents) on Wang resin (330 mg, 0.20 mmol). After complete deprotection and cleavage from the resin with the cleavage cocktail TFA/DCM/TIS = 47 : 47 : 6 during 2 h, (protocol 10) the cyclic peptide (45) is purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (45)): 13 mg, 30 µmol, 15 %.

HPLC (anal. method 2): tR=16.4 min, 100 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 640.33 [M+H]⁺, 662.31 [M+Na]⁺, 678.29 [M+K]⁺ Found: 640.74 [M+H]⁺, 662.73 [M+Na]⁺, 678.69 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two sets of signals (ratio 6 : 1).

0.77 (d, ³J=6.3 Hz, 3H, H^γ (D-Val)), 0.79 (d, ³J=6.3 Hz, 3H, H^δ (Leu)), 0.85 (d,

3J=6.9 Hz, 3H, H^δ(Leu)), 0.92 (d, ³J=6.9 Hz, 3H, H^γ(D-Val)), 1.34 (ddd, ²J=13.7 Hz, ³J=11.5 Hz, ³J=3.0 Hz, 1H H^β(Leu)), 1.48-1.59 (m, 2H, H^γ(Leu), H^γ(β-hPro)), 1.67 (ddd, ²J=13.7 Hz, ³J=10.2 Hz, ³J=3.3 Hz, 1H, H^β(Leu)), 1.73-1.87 (m, 3H, 2H^δ (β-hPro), H^β(D-Val)), 2.04 (dddd, ²J=12.1 Hz, ³J=6.1 Hz, ³J=6.1 Hz, ³J=6.1 Hz, 1H, H^γ (β-hPro)), 2.15 (d, ²J=14.5 Hz, 1H, H^α(β-hPro)), 2.25 (dd, ²J=15.1 Hz, ³J=7.5 Hz, 1H, H^α(β-hPro)), 2.34-2.47 (m, 3H, H^β(Asx)), 2.79 (dd, ²J=16.6 Hz, ³J=3.5 Hz, 1H, H^β(Asx)), 3.50 (dd, ³J=9.4 Hz, ³J=5.0 Hz, 1H, H^α(D-Val)), 3.55-3.62 (m, 2H, H^ε(β-hPro), H^β(Ser)), 3.70 (ddd, ²J=9.7 Hz, ³J=7.5 Hz, ³J=4.7 Hz, 1H, H^ε(β-hPro)), 3.83 (ddd, ²J=11.0 Hz, ³J=6.9 Hz, ³J=6.9 Hz, 1H, H^β (Ser)), 4.06 (m, 1H, H^β (β-hPro)), 4.25 (ddd, ³J=11.5 Hz, ³J=8.3 Hz, ³J=3.3 Hz, 1H, H^α(Leu)), 4.49 (ddd,

3J=9.7 Hz, ³J=9.0 Hz, ³J=3.5 Hz, 1H, H^α(Asx)), 4.68 (ddd, ³J=9.1 Hz, ³J=7.2 Hz,

3J=7.2 Hz, 1H, H^α (Ser)), 4.74 (ddd, ³J=9.3 Hz, ³J=6.7 Hz, ³J=4.6 Hz, 1H, H^α (Asx)), 4.80 (dd, ³J=6.3 Hz, ³J=6.3 Hz, 1H, H^O (Ser)), 6.84 (s, 1H, H^N (Asn)), 7.23 (s, 1H, H^N (Asn)), 7.74 (d, ³J=6.3 Hz, 1H, H^N (Asx)), 7.66 (d, ³J=8.2 Hz, 1H, H^N (Leu)), 7.95 (d, ³J=8.8 Hz, 1H, H^N (Ser)), 8.50 (d, ³J=5.0 Hz, 1H, H^N (D-Val)), 8.60 (d, ³J=8.8 Hz, 1H, H^N (Asx)), 12.29 (br, 1H, H^COO (Asp)).

c-(-Asp-Ser-Pro-Leu-Asn-) (46)

C22H34N6O9 (526.55 g/mol).

Cyclization on resin:

The linear peptide H-Ser(t-Bu)-Pro-Leu-Asn(Trt)-Asp(Wang)-ODmb (87) is synthesized and cyclized following protocol 6 on Wang resin. After complete deprotection and cleavage from the resin (Table 35, protocol 10) cyclic peptide (46)

is purified by reverse phase preparative HPLC.

Table 35: On resin cyclization of H-Ser(t-Bu)-Pro-Leu-Asn(Trt)-Asp(Wang)-ODmb (87).

Resin n/

mmol Coupling reagent t/

min

Cleavage Reagent

t/

h Yield HPLC/

% Wang

135 mg 0.10 1.1 equiv HATU

3.0 equiv DIPEA 45 Reagent

B 5

2 mg 2 µmol

2 %

57^a

Wang

130 mg 0.10 3.0 equiv HATU

6.0 equiv DIPEA 2x45

TFA 47 DCM 47

TIS 6

2

13mg 22 µmol

22 %

90^a

a The same HPLC analytical method and the retention time as for the cyclization in solution.

Cyclization in solution:

The linear peptide H-Leu-Asn(Trt)-Asp(Ot-Bu)-Ser(t-Bu)-Pro-OH (88) is synthesized on 2-ClTrt resin (240 mg 0.20 mmol) previously loaded with Fmoc- -Pro-OH (loading 0.84 mmol/g), and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (88)): 101 mg, 90 µmol, 45 %.

HPLC (anal. method 1): tR=27.8 min, 80 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 899.49 [M+H]⁺, 921.47 [M+Na]⁺, 937.45 [M+K]⁺ Found: 921.51 [M+Na]⁺, 937.53 [M+K]⁺

The linear peptide (88) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (88) (101 mg, 90 µmol) in 11 ml DMF.

Second syringe: Solution of HATU (1.5 equiv, 135 µmol, 51 mg) in 11 ml DMF.

Addition rate for both syringes: 0.01 ml/min.

Flask: Solution of DIPEA (3.0 equiv, 270 µmol, 35 mg, 46 µl) and HOAt (1.0 equiv, 90 µmol, 12 mg) in 20 ml DMF.

After evaporation of DMF the cyclic protected peptide (89) is purified by reverse phase preparative HPLC.

Yield (cyclic protected peptide (89)): 90 mg, 60 µmol, 68 %.

HPLC (anal. method 1): tR=31.3 min, 60 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 881.48 [M+H]⁺, 903.46 [M+Na]⁺, 919.44 [M+K]⁺ Found: 903.52 [M+Na]⁺, 919.55 [M+K]⁺

The cyclic protected peptide (89) is completely deprotected with reagent K during 5 h (protocol 13) and finally purified by preparative HPLC.

Yield (cyclic deprotected peptide (46)): 79 mg, 60 µmol, 100 %.

HPLC (anal. method 2): tR=15.0 min, 40 area %. No isomers detected.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 527.25 [M+H]⁺, 549.23 [M+Na]⁺, 565.20 [M+K]⁺ Found: 549.49 [M+Na]⁺, 565.47 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): 0.82 (d, ³J=6.3 Hz, 3H, H^δ(Leu)), 0.85 (d,

3J=6.3 Hz, 3H, H^δ(Leu)), 1.28-1.43 (m, 3H, 2H^β(Leu), H^γ(Leu)), 1.66 (m, 1H, H^γ (Pro)), 1.81-1.93 (m, 2H, H^β(Pro), H^γ(Pro)), 2.10 (dd, ²J=15.7 Hz, ³J=4.4 Hz, 1H, H^β (Asx)), 2.19 (m, 1H, H^β (Pro)), 2.35 (m, 1H, H^β (Asx)), 2.38 (dd, ²J=12.2 Hz,

3J=9.7 Hz, 1H, H^β(Asx)), 2.58 (dd, ²J=15.4 Hz, ³J=9.1 Hz, 1H, H^β(Asx)), 3.40-3.50 (m, 3H, H^δ(Pro), 2H^β(Ser)), 3.55 (ddd, ²J=11.2 Hz, ³J=8.0 Hz, ³J=3.3 Hz, 1H, H^δ (Pro)), 4.12 (ddd, ³J=9.7 Hz, ³J=9.7 Hz, ³J=3.5 Hz, 1H, H^α(Asx)), 4.34 (ddd, ³J=9.1 Hz, ³J=9.1 Hz, ³J=6.3 Hz, 1H, H^α(Leu)), 4.47 (ddd, ³J=8.6 Hz, ³J=4.9 Hz, ³J=3.9 Hz, 1H, H^α(Ser)), 4.51 (dd, ³J=8.8 Hz, ³J=2.5 Hz, 1H, H^α(Pro)), 4.83 (ddd, ³J=9.1 Hz, ³J=9.1 Hz, ³J=4.1Hz, 1H, H^α (Asx)), 4.95 (dd, ³J=6.3 Hz, ³J=5.0 Hz, 1H, H^O (Ser)), 6.72 (s, 1H, H^N (Asn)), 7.01 (d, ³J=8.8 Hz, 1H, H^N (Ser)), 7.24 (s, 1H, H^N (Asn)), 7.39 (d, ³J=9.4 Hz, 1H, H^N (Leu)), 7.75 (d, ³J=10.0 Hz, 1H, H^N (Asx)), 8.35 (d, ³J=8.8 Hz, 1H, H^N (Asx)), 12.5 (br, 1H, H^COO (Asp)).

c-(-Asp-Ser-Pro-Leu-ββββ-hAsn-) (47)

C23H36N6O9 (540.58 g/mol).

Cyclization in solution:

The linear peptide H-β-hAsn(Trt)-Asp(Ot-Bu)-Ser(t-Bu)-Pro-Leu-OH (90) is synthesized on 280 mg (0.20 mmol) Fmoc-Leu-Sasrin (loading 0.72 mmol/g) and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (90)): 167 mg, 0.15 mmol, 75 %.

HPLC (anal. method 2): tR=29.5 min, 82 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 913.51 [M+H]⁺, 935.49 [M+Na]⁺, 951.46 [M+K]⁺ Found: 913.33 [M+H]⁺, 935.45 [M+Na]⁺, 951.39 [M+K]⁺ The linear peptide (90) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (90) (180 mg, 0.14 mmol) in 9 ml DMF.

Second syringe: Solution of HATU (1.5 equiv, 0.22 mmol, 84mg) in 9 ml DMF.

Addition rate for both syringes: 0.02 ml/min.

Flask: Solution of DIPEA (6.0 equiv, 0.90 mmol, 116 mg, 154 µl) and HATU (0.1 equiv, 15 µmol, 6 mg) in 20 ml DMF.

After evaporation of DMF the cyclic protected peptide (91) is purified by reverse phase preparative HPLC.

Yield (cyclic protected peptide (91)): 94 mg, 0.11 mmol, 70. %.

HPLC (anal. method 1): tR=31.8 min, 100 area % sum of epimers.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 895.50 [M+H]⁺, 917.48 [M+Na]⁺, 933.45 [M+K]⁺ Found: 917.57 [M+Na]⁺, 933.74 [M+K]⁺

The cyclic protected peptide (91) is completely deprotected with a cleavage cocktail TFA/H2O/TIS = 95 : 2.5 : 2.5 during 2 h (protocol 13) and finally purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (47)): 25 mg, 46 µmol, 44 %.

HPLC (anal. method 2): tR=13.0 min, 13.6 min, 100 area %, epimers ratio 40:60.

For the biological tests and the NMR analysis the epimers were separated by reverse phase preparative HPLC (preparative method 3).

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 541.26 [M+H]⁺, 563.24 [M+Na]⁺, 579.22 [M+K]⁺ Found: 541.27 [M+H]⁺, 563.28 [M+Na]⁺, 579.47 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): 0.82 (d, ³J=6.3 Hz, 3H, H^δ(Leu)), 0.89 (d,

3J=6.3 Hz, 3H, H^δ(Leu)), 1.37-1.63 (m, 4H, H^β(Leu), H^γ(Leu), 2H^γ(Pro)), 1.86-1.98 (m, 2H, H^β(Leu), H^β(Pro)), 2.15 (dd, ²J=13.2 Hz, ³J=3.1 Hz, 1H, H^γ (β-hAsn)), 2.19

(m, 1H, H^β (Pro)), 2.28 (dd, ²J=14.4 Hz, ³J=6.9 Hz, 1H, H^α (β-hAsn)), 2.35 (dd,

2J=14.4 Hz, ³J=5.7 Hz, 1H, H^α (β-hAsn)), 2.42-2.46 (m, 2H, H^β (Asp)), 2.59 (dd,

2J=13.2 Hz, ³J=11.9 Hz 1H, H^γ (β-hAsn)), 3.31 (ddd, ²J=11.8 Hz, ³J=8.9 Hz, ³J=2.4 Hz, 1H, H^δ (Pro)), 3.34-3.51 (m, 2H, H^δ (Pro), H^β (Ser)), 3.62 (dd, ²J=10.1 Hz,

3J=5.7 Hz, 1H, H^β (Ser)), 4.09-4.19 (m, 2H, H^β (β-hAsn), H^α (Ser)), 4.32 (ddd,

3J=9.6 Hz, ³J=9.6 Hz, ³J=4.9 Hz, 1H, H^α(Leu)), 4.55 (ddd, ³J=8.9 Hz, ³J=7.7 Hz,

3J=6.8 Hz, 1H, H^α (Asp)), 4.64 (dd, ³J=8.2 Hz, ³J=1.0 Hz, 1H, H^α(Pro)), 6.82 (s, 1H, H^N (β-hAsn)), 7.31 (s, 1H, H^N (β-hAsn)), 7.52 (d, ³J=7.5 Hz, 1H, H^N (β-hAsn)), 7.96 (d, ³J=8.8 Hz, 1H, H^N (Asp)), 8.04-8.09 (m, 2H, H^N (Leu), H^N (Ser))

c-(-ββββ-hAsp-Pro-Leu-Asn-) (48)

C20H31N5O7 (453.50 g/mol).

Cyclization in solution:

The linear peptide H-Asn(Trt)-β-hAsp(Ot-Bu)-Pro-Leu-OH (92) is synthesized on 2-ClTrt resin (280 mg, 0.20 mmol) previously loaded with Fmoc-Leu-OH (loading 0.72 mmol/g), and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (92)): 129 mg, 0.14 mmol, 68 %.

HPLC (anal. method 1): tR=26.5 min, 80 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 770.41 [M+H]⁺, 792.39 [M+Na]⁺, 808.37 [M+K]⁺ Found: 770.82 [M+Na]⁺, 792.81 [M+K]⁺

The linear peptide (92) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (92) (129 mg, 0.14 mmol) in 10 ml DMF.

Second syringe: Solution of HATU (3.9 equiv, 0.53 mmol, 200 mg) in 10 ml DMF.

Addition rate for both syringes: 0.01 ml/min.

Flask: Solution of DIPEA (8.5 equiv, 1.2 mmol, 151 mg, 200 µl) in 10 ml DMF.

After evaporation of DMF cyclic protected peptide (93) is purified by reverse phase preparative HPLC.

Yield (cyclic protected peptide (93)): 51 mg, 48 µmol, 35 %.

HPLC (anal. method 1): tR=30.5 min, 70 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 752.40 [M+H]⁺, 774.38 [M+Na]⁺, 790.36 [M+K]⁺ Found: 774.38 [M+Na]⁺, 790.48 [M+K]⁺

The cyclic protected peptide (93) is completely deprotected with reagent K during 5 h (protocol 13) and finally purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (48)): 16 mg, 28 µmol, 58 %.

HPLC (anal. method 2): tR=16.5 min, 82 area %. No isomers detected.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 454.23 [M+H]⁺, 476.21 [M+Na]⁺, 492.19 [M+K]⁺ Found: 454.62 [M+H]⁺, 476.61 [M+Na]⁺, 492.57 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): 0.85 (d, ³J=6.3 Hz, 3H, H^δ(Leu)), 0.88 (d,

3J=6.9 Hz, 3H, H^δ(Leu)), 1.36-1.45 (m, 2H, H^β(Leu), H^γ(Leu)), 1.49 (ddd, ²J=13.7 Hz, ³J=6.4 Hz, ³J=6.4 Hz, 1H, H^β(Leu)), 1.69 (dddd, ²J=12.7 Hz, ³J=6.3 Hz, ³J=6.3 Hz, ³J=6.3 Hz, 1H, H^β(Pro)), 1.85-1.92 (m, 2H, H^γ(Pro)), 2.17 (dddd, ²J=12.6 Hz,

3J=8.8 Hz, ³J=7.1 Hz, ³J=7.1 Hz, 1H, H^β(Pro)), 2.26 (dd, ²J=14.4 Hz, ³J=8.8 Hz, 1H, H^β(Asn)), 2.45 (dd, ²J=10.1 Hz, ³J=6.3 Hz, 1H, H^α(γ)(β-hAsp)), 2.47-2.52 (m, 2H, H^β(Asn), H^α(γ)(β-hAsp)), 2.56 (dd, ²J=16.0 Hz, ³J=3.5 Hz, 1H, H^α(γ)(β-hAsp)), 2.81 (dd, ²J=15.7 Hz, ³J=12.6 Hz, 1H, H^α(γ)(β-hAsp)), 3.47 (ddd, ²J=9.4 Hz, ³J=6.9 Hz, ³J=6.9 Hz, 1H, H^δ(Pro)), 3.77 (ddd, ²J=9.7 Hz, ³J=6.6 Hz, ³J=6.6 Hz, 1H, H^δ (Pro)), 4.09 (dd, ³J=8.8 Hz, ³J=5.8 Hz 1H, H^α (Pro)), 4.14 (m, 1H, H^β(β-hAsp)), 4.36 (ddd, ³J=10.1 Hz, ³J=9.7 Hz, ³J=6.9 Hz, 1H, H^α(Leu)), 4.44 (ddd, ³J=9.1 Hz,

3J=9.1 Hz, ³J=5.7 Hz, 1H, H^α(Asn)), 6.95 (s, 1H, H^N (Asn)), 7.11 (d, ³J=10.2 Hz, 1H, H^N (Leu)), 7.14 (d, ³J=6.9 Hz, 1H, H^N (β-hAsp)), 7.49 (s, 1H, H^N (Asn)), 7.66 (d,

3J=10.1 Hz, 1H, H^N (Asn)), 12.31 (br, 1H, H^coo (β-hAsp)).

c-(-Thr-Gln-Ile-Asp-Ser-Pro-) (49)

C27H43N7O11 (641.69 g/mol).

Cyclization on resin:

The linear peptide H-Ser(t-Bu)-Pro-Thr(t-Bu)-Gln(Trt)-Ile-Asp(Wang)-ODmb (94) is synthesized and cyclized following protocol 6, using different coupling reagents (Table 36), on Wang resin. After complete deprotection and cleavage from the resin with different cleavage cocktails (Table 36) the cyclic peptide (49) is purified by reverse phase preparative HPLC.

Table 36: On resin cyclization of H-Ser(t-Bu)-Pro-Thr(t-Bu)-Gln(Trt)-Ile-Asp(Wang)-ODmb (94)

Resin n/

mmol Coupling reagent t/

min

Cleavage Reagent

t/

h Yield HPLC /%

Wang

300 mg 0.20 3.0 equiv HATU

6.0 equiv DIPEA 45 Reagent K 5 0 0 Wang

130 mg 0.10 2.0 equiv PyBOP

6.0 equiv DIPEA 60 Reagent K 2 0 0 Wang

130 mg 0.1 1.1 equiv PyAOP

3.0 equiv DIPEA 2x30

TFA 95 % H2O 2.5 % TIS 2.5 %

2 0 0

Wang

500 mg 0.2 3.0 equiv HATU 6.0 equiv DIPEA 45

TFA 95 % H2O 2.5 % TIS 2.5 %

2

12 mg 15 µmol

8 %

80^a

a The same HPLC analytical method and the retention time as for the cyclization in solution.

Cyclization in solution:

The linear peptide H-lle-Asp(Ot-Bu)-Ser(t-Bu)-Pro-Thr(t-Bu)-Gln(Trt)-OH (95) is synthesized on 2-ClTrt resin (300 mg, 0.20 mmol) previously loaded with Fmoc-Gln(Trt)-OH (loading 0.67 mmol/g), and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (95)): 209 mg, 0.12 mmol, 58 %.

HPLC (anal. method 1): tR=29.5 min, 60 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1070.62 [M+H]⁺, 1092.60 [M+Na]⁺, 1108.57 [M+K]⁺ Found: 1092.82 [M+Na]⁺, 1108.76 [M+K]⁺

The linear peptide (95) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (95) (209 mg, 0.12 mmol) in 10 ml DMF.

Second syringe: Solution of HATU (4.5 equiv, 0.53 mmol, 200 mg) in 10 ml DMF.

Addition rate for both syringes: 0.01 ml/min.

Flask: Solution of DIPEA (10.0 equiv, 1.17 mmol, 151 mg, 200 µl) in 20 ml DMF.

After cyclization, DMF is evaporated and the protected cyclic peptide (96) is purified by filtration through a C18 plug.

Yield (cyclic protected peptide (96)): 37 mg, 35 µmol, 30 %.

HPLC (anal. method 1): tR=35.5 min, 35.9 min, 100 area % sum of epimers.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1052.61 [M+H]⁺, 1074.59 [M+Na]⁺, 1090.56 [M+K]⁺ Found: 1074.56 [M+Na]⁺, 1090.81 [M+K]⁺

A similar procedure is used for the synthesis of the same cyclic peptide (96) from different linear precursors (97)-(99). Results are shown in Table 15, P. 68.

The cyclic protected peptide (96) is complete deprotected with reagent K during 2 h (protocol 13) and finally purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (49)): 11 mg, 17 µmol, 49 %.

HPLC (anal. method 2): tR=12.0 min, 13.3 min, 100 area %, epimers ratio 37:63.

For the biological tests and the NMR analysis the epimers were separated by reverse phase preparative HPLC (preparative method 3).

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 642.31 [M+H]⁺, 664.29 [M+Na]⁺, 680.27 [M+K]⁺ Found: 642.57 [M+H]⁺, 664.68 [M+Na]⁺, 680.58 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two sets of signals (ratio 2 : 1).

Major conformer 0.79 (d, ³J=6.9 Hz, 3H, H^γ(Ile)), 0.80 (dd, ³J=7.5 Hz, ³J=7.5 Hz, 3H, H^δ(Ile)), 1.06 (m, 1H, H^γ(Ile)), 1.15 (d, ³J=5.7 Hz, 3H, H^γ(Thr)), 1.32 (m, 1H, H^γ(Ile)), 1.54 (m, 1H, H^γ(Pro)), 1.70-1.83 (m, 2H, H^β(Ile), H^β(Pro)), 1.83-1.91 (m, 2H, H^γ(Pro), H^β(Gln)), 2.06-2.12 (m, 2H, H^β(Gln), H^γ(Gln)), 2.17 (m, 1H, H^γ(Gln)), 2.25 (m, 1H, H^β(Pro)), 2.36 (dd, ²J=16.0 Hz, ³J=7.9 Hz, 1H, H^β(Asp)), 2.54 (dd,

2J=15.9 Hz, ³J=5.7 Hz, 1H, H^β (Asp)), 3.40 (br, 1H, H^δ(Pro)), 3.45 (ddd, ²J=11.3 Hz, ³J=11.3 Hz, ³J=6.6 Hz, 1H, H^δ(Pro)), 3.54 (m, 1H, H^β(Ser)), 3.66 (dd, ²J=10.1 Hz, ³J=5.7 Hz, 1H, H^β (Ser)), 3.81 (dd, ³J=7.5 Hz, ³J=4.4 Hz, 1H, H^α(Ile)), 4.16 (dd, ³J=10.1 Hz, ³J=2.5 Hz, 1H, H^α(Pro)), 4.28-4.38 (m, 2H, H^α(Gln), H^α(Ser)), 4.48-4.58 (m, 3H, H^α(Asp), H^α(Thr), H^β(Thr)), 5.04 (br, 1H, H^O), 5.38 (br, 1H, H^O), 6.77 (d, ³J=6.3 Hz, 1H, H^N (Asp)), 6.87 (s, 1H, H^N (Gln)), 7.25 (m, 1H, H^N (Ser)), 7.37 (s, 1H, H^N (Gln)), 8.47 (d, ³J=4.4 Hz, 1H, H^N (Ile)), 8.74 (d, ³J=10.1 Hz, 1H, H^N (Thr)), 8.84 (br, 1H, H^N (Gln)), 12.32 (br, 1H, H^COO (Asp)).

Minor conformer 0.83 (d, ³J=6.9 Hz, 3H, H^γ(Ile)), 0.85 (dd, ³J=7.5 Hz, ³J=7.5 Hz, 3H, H^δ(Ile)), 0.97 (d, ³J=6.3 Hz, 3H, H^γ(Thr)), 1.06 (m, 1H, H^γ(Ile)), 1.32 (m, 1H, H^γ(Ile)), 1.76 (m, 1H, H^β(Ile)), 1.82-1.91 (m, 3H, 2H^β(Gln), H^γ(Pro)), 1.90-2.03 (m, 3H, 2H^β(Pro), H^γ(Pro)), 2.22-2.31 (m, 2H, H^γ(Gln)), 2.65 (dd, ²J=16.3 Hz, ³J=8.2 Hz, 1H, H^β(Asp)), 2.92 (dd, ²J=16.3 Hz, ³J=5.0 Hz, 1H, H^β(Asp)), 3.54 (m, 1H, H^β (Ser)), 3.61 (ddd, ²J=10.7 Hz, ³J=9.7 Hz, ³J=3.1 Hz, 1H, H^δ(Pro)), 3.77-3.84 (m, 2H, H^α(Gln), H^δ(Pro)), 3.97 (dd, ³J=8.8 Hz, ³J=8.2 Hz, 1H, H^α(Ile)), 3.91 (m, 1H,

H^β(Ser)), 4.18-4.25 (m, 2H, H^α(Thr), H^β(Thr)), 4.33 (m, 1H, H^α(Asp)), 4.69-4.76 (m, 2H, H^α(Pro), H^α(Ser)), 4.93 (br, 1H, H^O), 5.38 (br, 1H, H^O), 6.88 (s, 1H, H^N (Gln)), 7.21-7.30 (m, 2H, H^N (Gln), H^N (Ile)), 7.49 (d, ³J=8.8 Hz, 1H, H^N (Thr)), 7.77 (d, ³J=6.9 Hz, 1H, H^N (Ser)), 8.19 (d, ³J=5.0 Hz, 1H, H^N (Gln)), 8.59 (br, 1H, H^N (Asp)), 12.32 (br, 1H, H^COO (Asp)).

c-(-Thr-Gln-Ile-Asp-Ser-ββββ-hPro-) (50)

C28H45N7O11 (655.71 g/mol).

Cyclization on resin:

The linear peptide H-Ser(t-Bu)-β-hPro-Thr(t-Bu)-Gln(Trt)-Ile-Asp(Wang)-ODmb

(100) is synthesized and cyclized (3 equiv HATU and 6 equiv DIPEA as coupling reagents), on Wang resin (400 mg, 0.20 mmol) following protocol 6. After cleavage from the resin and deprotection (protocol 10), the cyclic peptide (50) is purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (50)): 6 mg, 8 µmol, 4 %.

HPLC (anal. method 2): tR=13.4 min, 90 area %.

Cyclization in solution:

The linear peptide H-lle-Asp(Ot-Bu)-Ser(t-Bu)-β-hPro-Thr(t-Bu)-Gln(Trt)-OH (101) is synthesized on 2-ClTrt resin (300 mg, 0.20 mmol) previously loaded with Fmoc-Gln(Trt)-OH (loading 0.67 mmol/g), and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (101)): 190 mg, 0.15 mmol, 74 %.

HPLC (anal. method 1): tR=29.2 min, 84 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1084.63 [M+H]⁺, 1106.62 [M+Na]⁺, 1122.59 [M+K]⁺ Found: 1084.82 [M+H]⁺, 1106.83 [M+Na]⁺, 1122.69 [M+K]⁺ The linear peptide (101) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (101) (190 mg, 0.15 mmol) in 15 ml DMF.

Second syringe: Solution of HATU (1.8 equiv, 0.26 mmol, 100 mg) in 15 ml DMF.

Addition rate for both syringes: 0.01 ml/min.

Flask: Solution of DIPEA (6.0 equiv, 0.88 mmol, 113 mg, 150 µl) and HOAt (1.0 equiv, 0.15 mmol, 20 mg) in 20 ml DMF.

After evaporation of DMF the cyclic protected peptide (102) is purified by reverse phase preparative HPLC.

Yield (cyclic protected peptide (102)): 114 mg, 0.11 mmol, 73 %.

HPLC (anal. method 1): tR=35.8 min, 36.4 min, 100 area % sum of epimers.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1066.62 [M+H]⁺, 1088.60 [M+Na]⁺, 1104.58 [M+K]⁺ Found: 1088.52 [M+Na]⁺, 1104.88 [M+K]⁺

The cyclic protected peptide (102) is completely deprotected with reagent K during 5 h (protocol 13) and finally purified by preparative HPLC.

Yield (cyclic deprotected peptide (50)): 36 mg, 55 µmol, 51 %.

HPLC (anal. method 2): tR=12.4 min, 13.7 min, 100 area %, epimers ratio 43:57.

For the biological tests and the NMR analysis the epimers were separated by reverse phase preparative HPLC (preparative method 3).

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 656.32 [M+H]⁺, 678.31 [M+Na]⁺, 694.28 [M+K]⁺ Found: 656.37 [M+H]⁺, 678.38 [M+Na]⁺, 694.29 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two sets of signals (ratio 2 : 1).

Major conformer 0.80 (dd, ³J=7.2 Hz, ³J=7.2 Hz, 3H, H^γ(Ile)), 0.82 (d, ³J=6.3 Hz, 3H, H^δ(Ile)), 0.99 (d, ³J=6.3 Hz, 3H, H^γ(Thr)), 1.18 (m, 1H, H^γ(Ile)), 1.30 (m, 1H, H^γ(Ile)), 1.67-2.15 (m, 8H, 2H^β(Gln), H^γ(Gln), H^β(Ile), 2H^γ(β-hPro), 2H^δ(β-hPro)), 2.43-2.53 (br, 2H, H^γ(Gln), H^α(β-hPro)), 2.61 (dd, ²J=17.3 Hz, ³J=9.1 Hz, 1H, H^β (Asp)), 2.73 (dd, ²J=17.3 Hz, ³J=3.5 Hz, 1H, H^β(Asp)), 2.93 (m, 1H, H^α(β-hPro)), 3.26 (ddd, ²J=11.3 Hz, ³J=8.2 Hz, ³J=3.5 Hz, 1H, H^ε(β-hPro)), 3.20-3.45 (br, 2H, H^ε(β-hPro), H^β(Ser)), 3.56 (dd, ²J=10.1 Hz, ³J=8.1 Hz, 1H, H^β(Ser)), 3.85 (m, 1H, H^β (Thr)), 4.08 (dd, ³J=8.8 Hz, ³J=4.4 Hz, 1H, H^α (Ile)), 4.26 (ddd, ³J=6.8 Hz,

3J=6.8 Hz, ³J=6.8 Hz, 1H, H^α(Gln)), 4.31 (dd, ³J=7.9 Hz, ³J=4.7 Hz, 1H, H^α(Thr)), 4.39 (m, 1H, H^β (β-hPro)), 4.55 (ddd, ³J=7.9 Hz, ³J=7.2 Hz, ³J=6.6 Hz, 1H, H^α (Ser)), 4.74 (ddd, ³J=9.3 Hz, ³J=9.3 Hz, ³J=3.3 Hz, 1H, H^α(Asp)), 6.74 (s, 1H, H^N (Gln)), 7.28 (s, 1H, H^N (Gln)), 7.42 (d, ³J=8.2 Hz, 1H, H^N (Thr)), 7.50 (d, ³J=9.4 Hz, 1H, H^N (Asp)), 8.1 (d, ³J=8.8 Hz, 1H, H^N (Ile)), 8.35 (d, ³J=6.9 Hz, 1H, H^N (Ser)), 8.46 (d, ³J=6.9 Hz, 1H, H^N (Gln)), 12.30 (br, 1H, H^COO (Asp)).

Minor conformer 0.78 (dd, ³J=6.9 Hz, ³J=6.9 Hz, 3H, H^γ(Ile)), 0.82 (d, ³J=6.3 Hz, 3H, H^δ(Ile)), 1.02 (d, ³J=6.3 Hz, 3H, H^γ(Thr)), 1.05 (m, 1H, H^γ(Ile)), 1.39 (m, 1H, H^γ(Ile)), 1.67-2.15 (m, 8H, 2H^β(Gln), H^γ(Gln), H^β(Ile), 2H^γ(β-hPro), 2H^δ(β-hPro)), 2.25 (dd, ²J=14.1 Hz, ³J=1.0 Hz, 1H, H^α(β-hPro)), 2.43-2.53 (br, 3H, H^β(Asp), H^γ (Gln), H^α(β-hPro)), 2.93 (dd, ²J=16.3 Hz, ³J=5.7 Hz, 1H, H^β(Asp)), 3.20-3.45 (br, 3H, 2H^ε(β-hPro), H^β(Ser)), 3.49 (m, 1H, H^β(Ser)), 3.90 (dd, ³J=9.7 Hz, ³J=8.5 Hz, 1H, H^α(Ile)), 3.95 (m, 1H, H^β(Thr)), 4.08 (m, 1H, H^α(Asp)), 4.15 (dd, ³J=7.5 Hz,

3J=3.8 Hz, 1H, H^α(Thr)), 4.39 (m, 1H, H^β(β-hPro)), 4.44 (ddd, ³J= 8.6 Hz, ³J=4.6 Hz, ³J=4.6 Hz, 1H, H^α(Gln)), 4.61 (ddd, ³J= 8.5 Hz, ³J=6.3 Hz, ³J=6.3 Hz, 1H, H^α (Ser)), 6.71 (s, 1H, H^N (Gln)), 6.73 (d, ³J=8.8 Hz, 1H, H^N (Ser)), 7.14 (s, 1H, H^N

(Gln)), 7.29 (d, ³J=7.5 Hz, 1H, H^N (Thr)), 7.98 (d, ³J=8.8 Hz, 1H, H^N (Gln)), 8.12 (d,

3J=8.2 Hz, 1H, H^N (Ile)), 8.80 (d, ³J=7.5 Hz, 1H, H^N (Asp)), 12.30 (br, 1H, H^COO (Asp)).

c-(-Thr-ββββ-hGln-Ile-Asp-ββββ-hSer-Pro-) (51)

C29H47N7O11 (669.74 g/mol).

Cyclization on resin:

The linear peptide H-β-hSer(t-Bu)-Pro-Thr(t-Bu)- β-hGln-Ile-Asp(Wang)-ODmb

(103) is synthesized and cyclized following protocol 6 (3.0 equiv HATU and 6.0 equiv DIPEA as coupling reagents), on Wang resin (200 mg, 92 µmol). After complete deprotection and cleavage from the resin with the cleavage cocktail TFA/DCM/TIS = 47 : 47: 6 during 2 h (protocol 10) the cyclic peptide (51) is purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (51)): 3 mg, 5 µmol, 5 %.

HPLC (anal. method 2): tR=15.3 min, 100 area %.

Cyclization in solution:

a) The linear peptide H-β-hGln-Ile-Asp(Ot-Bu)-β-hSer(t-Bu)-Pro-Thr(t-Bu)-OH (104)

is synthesized on 2-ClTrt resin (125 mg, 0.10 mmol) previously loaded with Fmoc-Thr(t-Bu)-OH (loading 0.80 mmol/g), and cleaved with 1 % TFA in DCM (protocol 9). The linear protected peptide (104) is not purified.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 856.54 [M+H]⁺, 878.52 [M+Na]⁺, 894.50 [M+K]⁺ Found: 856.74 [M+H]⁺, 878.55 [M+Na]⁺, 894.62 [M+K]⁺ The linear peptide (104), is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (104) in 7 ml DMF.

Second syringe: Solution of HATU (110 µmol, 42 mg) in 7 ml DMF.

Addition rate for both syringes: 0.005 ml/min.

Flask: Solution of DIPEA (300 µmol, 39 mg, 51 µl) and HATU (50 µmol, 19 mg) in 10 ml DMF.

After evaporation of the solvent the cyclic protected peptide (105) is purified by reverse phase preparative HPLC.

Yield (cyclic protected peptide (105)): 15 mg, 18 µmol, 18 % (Calculated on resin loading).

HPLC (anal. method 1): tR=23.7 min, 100 area % sum of epimers.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 838.53 [M+H]⁺, 860.51 [M+Na]⁺, 876.48 [M+K]⁺ Found: 860.83 [M+Na]⁺, 876.83 [M+K]⁺

The cyclic protected peptide (105) is completely deprotected with the cleavage mixture TFA/H2O/TIS = 95 : 2.5 : 2.5 during 2 h (protocol 13) and finally purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (51)): 4 mg, 5.5 µmol, 31 %.

HPLC (anal. method 2): tR=13.6 min, 14.1 min, 92 area %, epimers ratio 1:1.

b) The linear peptide H-Thr(t-Bu)-β-hGln-Ile-Asp(Ot-Bu)-β-hSer(t-Bu)-Pro-OH (106)

is synthesized on 2-ClTrt resin (265 mg, 0.20 mmol) previously loaded with Fmoc-Pro-OH (loading 0.76 mmol/g), and cleaved with 1 % TFA in DCM (protocol 9). The linear protected peptide (106) is purified by filtration through a C18 plug.

Yield (linear protected peptide (106): 180 mg, 174 µmol, 87 %.

HPLC (anal. method 1): tR=22.6 min, 83 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 856.54 [M+H]⁺, 878.52 [M+Na]⁺, 894.50 [M+K]⁺ Found: 856.78 [M+H]⁺, 878.59 [M+Na]⁺, 894.60 [M+K]⁺ The linear peptide (106), is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (106) (180 mg, 174 µmol) in 20 ml DMF.

Second syringe: Solution of HATU (2.3 equiv, 0.4 mmol, 152 mg) in 20 ml DMF.

Addition rate for both syringes: 0.01 ml/min.

Flask: Solution of DIPEA (4.5 equiv, 0.8 mmol, 103 mg, 136 µl) and HOAt (2.3 equiv, 0.4 mmol, 54 mg) in 20 ml DMF.

After evaporation of the solvent the cyclic protected peptide (105) is purified by filtration through C18 plug.

Yield (cyclic protected peptide (105)): 92 mg, 66 µmol, 38 %.

HPLC (anal. method 1): tR=23.7 min, 60 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 838.53 [M+H]⁺, 860.51 [M+Na]⁺, 876.48 [M+K]⁺ Found: 860.81 [M+Na]⁺, 876.83 [M+K]⁺

The cyclic protected peptide (105) is completely deprotected with reagent K during 5 h, (protocol 13) and finally purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (51)): 14 mg, 21 µmol, 32 %.

HPLC (anal. method 2): tR=15.3 min, 100 area %. No isomers detected.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 670.34 [M+H]⁺, 692.32 [M+Na]⁺, 708.30 [M+K]⁺ Found: 670.43 [M+H]⁺, 692.45 [M+Na]⁺, 708.39 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): two sets of signals (ratio 3 :1).

Major conformer 0.84 (dd, ³J=6.9 Hz, ³J=6.9 Hz, 3H, H^δ(Ile)), 0.86 (d, ³J=6.3 Hz, 3H, H^γ (Ile)), 1.00 (d, ³J=6.3 Hz, 3H, H^γ(Thr)), 1.22 (ddq, ²J=13.0 Hz, ³J=8.2 Hz,

3J=6.9 Hz, 1H, H^γ(Ile)), 1.42 (ddq, ²J=13.3 Hz, ³J=6.7 Hz, ³J=6.3 Hz, 1H, H^γ(Ile)), 1.56-1.78 (m, 4H, 2H^γ(β-hGln), H^β(Ile), H^γ (Pro)), 1.83 (m, 1H, H^γ(Pro)), 2.00-2.10 (m, 4H, 2H^δ(β-hGln), 2H^β(Pro)), 2.32 (dd, ²J=15.1 Hz, ³J=3.1 Hz, 1H, H^α(β-hGln)), 2.37 (dd, ²J=16.9 Hz, ³J=3.8 Hz, 1H, H^α(β-hSer)), 2.58 (dd, ²J=15.1 Hz, ³J=6.9 Hz, 1H, H^α(β-hGln)), 2.64 (dd, ²J=16.6 Hz, ³J=9.7 Hz, 1H, H^β(Asp)), 2.77 (dd, ²J=17.0 Hz, ³J=3.5 Hz, 1H, H^β(Asp)), 2.80 (dd, ²J=16.6 Hz, ³J=11.6 Hz, 1H, H^α(β-hSer)), 3.18-3.66 (br, 4H, 2H^δ(Pro), 2H^γ(β-hSer)), 3.69 (dd, ³J=4.4 Hz, ³J=4.4 Hz, 1H, H^α (Ile)), 3.82 (m, 1H, H^β(β-hGln), 3.94 (dd, ³J=7.9 Hz, ³J=4.1 Hz, 1H, H^α(Thr)), 4.07-4.15 (m, 2H, H^β(β-hSer), H^β(Thr)), 4.38 (ddd, ³J=10.2 Hz, ³J=7.1 Hz, ³J=3.3 Hz, 1H, H^α(Asp)), 4.44 (dd, ³J=5.0 Hz, ³J=5.0 Hz, 1H, H^α(Pro)), 4.89-5.16 (br, 2H, H^O (β-hSer), H^O (Thr)), 6.85 (s, 1H, H^N (β-hGln)), 7.30 (d, ³J=9.4 Hz, 1H, H^N (β-hSer)), 7.31 (s, 1H, H^N (β-hGln)), 7.44 (d, ³J=7.5 Hz, 1H, H^N (Thr)), 7.69 (d, ³J=8.8 Hz, 1H, H^N (β-hGln)), 8.06 (d, ³J=6.9 Hz, 1H, H^N (Asp)), 8.22 (d, ³J=3.8 Hz, 1H, H^N (Ile)), 12.33 (br, 1H H^COO Asp).

Minor conformer 0.78-0.88 (m, 6H, 3H^γ (Ile), 3H^δ (Ile), 1.03 (d, ³J=6.3 Hz, 3H, H^γ (Thr)), 1.10 (m, 1H, H^γ (Ile)), 1.39 (m, 1H, H^γ (Ile)), 1.74 (m, 1H, H^β (Ile)), 1.58-1.71 (m, 2H, H^γ (Pro)), 1.81-1.91 (m, 3H, 2H^γ (β-hGln), H^β (Pro)), 2.12 (m, 1H, H^β (Pro)), 2.45 (m, 1H, H^α (β-hSer)), 2.47-2.53 (br, 2H, H^β (Asp)), 2.61-2.67 (m, 2H, H^δ (β-hGln)), 2.72-2.81 (m, 2H, H^α (β-hGln)), 2.93 (dd, ²J=16.0 Hz, ³J=7.2 Hz, 1H, H^α (β-hSer)), 3.00 (m, 1H, H^δ (Pro)), 3.18-3.66 (br, 3H, 2H^γ (β-hSer), H^δ (Pro)), 3.73-3.85 (m, 2H, H^β (β-hGln), H^β (β-hSer)), 3.94 (m 1H, H^α (Ile)), 4.03 (dd, ³J=8.6 Hz,

3J=2.5 Hz, 1H, H^α (Thr)), 4.20-4.28 (m, 2H, H^α (Pro), H^β (Thr)), 4.42 (m, 1H, H^α (Asp)), 4.89-5.16 (br, 2H, H^O (β-hSer), H^O (Thr)), 6.78 (s, 1H, H^N (β-hGln)), 7.08 (d,

3J=8.2 Hz, 1H, H^N (Thr), 7.18 (s, 1H, H^N (β-hGln)), 7.55-7.59 (m, 2H, H^N (Asp), H^N (β-hGln)), 7.77 (d, ³J=6.9 Hz, 1H, H^N (Ile)), 7.89 (d ³J=6.9 Hz, 1H, H^N (β-hSer)), 12.33 (br, 1H H^COO Asp).

c-(-Thr-Gln-Val-Asp-Ser-Pro-) (52)

C26H41N7O11 (627.66 g/mol).

Cyclization on resin:

The linear peptide H-Ser-Pro-Thr(t-Bu)-Gln(Trt)-Val-Asp(Wang)-ODmb (107) is synthesized and cyclized following protocol 6 (3.0 equiv HATU and 6.0 equiv DIPEA as coupling reagents), on Wang resin (330 mg, 0.2 mmol). After complete deprotection and cleavage from the resin with the cleavage cocktail TFA/DCM/TIS

= 48 : 48: 4 during 2 h (protocol 10), the cyclic peptide (52) is purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (52)): 6 mg, 10 µmol, 5 %.

HPLC (anal. method 2): tR=13.07 min, 100 area %.

Cyclization in solution:

The linear peptide H-Gln(Trt)-Val-Asp(Ot-Bu)-Ser(t-Bu)-Pro-Thr(t-Bu)-OH (108) is synthesized on 2-ClTrt resin (250 mg, 0.2 mmol) previously loaded with Fmoc-Thr(t-Bu)-OH (loading 0.80 mmol/g), and cleaved with 1 % TFA in DCM (protocol 9). The linear protected peptide (108) is neither purified by reverse phase preparative HPLC nor with filtration through C18 plug.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1056.60 [M+H]⁺, 1078.58 [M+Na]⁺, 1094.56 [M+K]⁺ Found: 1078.69 [M+Na]⁺, 1094.67 [M+K]⁺

The linear peptide (108) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (108) in 7 ml DMF.

Second syringe: Solution of HATU (0.22 mmol, 84 mg,) in 7 ml DMF.

Addition rate for both syringes: 0.01 ml/min.

Flask: Solution of DIPEA (0.66 mmol, 85 mg, 113 µl) and HATU (0.10 mmol, 37 mg) in 10 ml DMF.

After evaporation of the solvent the protected cyclic peptide (109) is purified by reverse phase preparative HPLC.

Yield (cyclic protected peptide (109)): 16 mg, 15 µmol, 8 % (calculated on resin loading).

HPLC (anal. method 1): tR=32.0 min, 100 area % sum of epimers.

Also 164 mg, 0.16 mmol of the linear peptide (108) is isolated by reverse phase preparative HPLC.

Repeated cyclization following protocol 7, method B:

First syringe: Solution of linear peptide (108) (164 mg, 0.16 mmol ) in 7 ml DMF.

Second syringe: Solution of HATU (2.0 equiv, 0.32 mmol, 117 mg,) in 7 ml DMF.

Addition rate for both syringes: 0.02 ml/min.

Flask: solution: DIPEA (6.0 equiv, 0.93 mmol, 120 mg, 159 µl) and HATU (1.0 equiv, 0.16 mmol, 59 mg) in 10 ml DMF.

After evaporation of the solvent the cyclic protected peptide (109) is purified by reverse phase preparative HPLC.

Yield (cyclic protected peptide (109)): 35 mg, 34 µmol, 22 %.

HPLC (anal. method 1): tR=32.0 min, 100 area % sum of epimers.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1038.59 [M+H]⁺, 1060.57 [M+Na]⁺, 1076.55 [M+K]⁺ Found: 1060.70 [M+Na]⁺, 1076.85 [M+K]⁺

In the reaction mixture linear protected peptide is again detected with MALDI-ToF MS.

The combined cyclic protected peptide (109) (51 mg, 49 µmol) is completely deprotected with the cleavage mixture TFA/H2O/TIS = 95 : 2.5 : 2.5 during 2 h (protocol 13) and finally purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (52)): 19 mg, 30 µmol, 61 %.

HPLC (anal. method 2): tR=13.1 min, 100 area %, sum of epimers.

For the biological tests and the NMR analysis the epimers were separated by reverse phase preparative HPLC (preparative method 3).

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 628.29 [M+H]⁺, 650.28 [M+Na]⁺, 666.25 [M+K]⁺ Found: 650.19 [M+Na]⁺, 666.26 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two sets of signals (ratio 1.5 : 1).

Major conformer 0.78 (d, ³J=6.3 Hz, 3H, H^γ(Val)), 0.82 (d, ³J=6.9 Hz, 3H, H^γ(Val)), 1.14 (d, ³J=6.3 Hz, 3H, H^γ (Thr)), 1.56 (m, 1H, H^γ (Pro)), 1.80-1.90 (m, 4H, 2H^β (Gln), H^β(Pro), H^γ(Pro)), 1.90-2.02 (m, 2H, H^β(Pro), H^β(Val)), 2.06-2.31 (m, 2H, H^γ (Gln)), 2.36 (dd, ²J=15.7 Hz, ³J=8.2 Hz, 1H, H^β(Asp)), 2.55 (dd, ²J=15.7 Hz, ³J=5.7 Hz, 1H, H^β(Asp)), 3.32-3.38 (m, 2H, 2H^δ(Pro)), 3.52 (br, 1H, H^β(Ser)), 3.65 (dd,

2J=10.4 Hz, ³J=5.3 Hz, 1H, H^β(Ser)), 3.80 (ddd, ³J=6.4 Hz, ³J=6.4 Hz, ³J=3.8 Hz, 1H, H^α(Gln)), 3.90 (m, 1H, H^α(Val)), 4.33 (m, 1H, H^α(Ser)), 4.47 (ddd, ³J=7.9 Hz,

3J=7.2 Hz, ³J=5.9 Hz, 1H, H^α(Asp)), 4.50-4.56 (m, 2H, H^α(Thr), H^β(Thr)), 4.69 (d,

3J=8.8 Hz, 1H, H^α(Pro)), 4.90-5.10 (br, 2H, H^O (Ser),H^O (Thr)), 6.73 (d, ³J=6.9 Hz, 1H, H^N (Asp)), 6.85 (s, 1H, H^N (Gln)), 7.20-7.26 (m, 2H, H^N (Ser), H^N (Val)), 7.45 (s, 1H, H^N (Gln)), 8.49 (d, ³J=3.8 Hz, 1H, H^N (Gln)), 8.71 (d, ³J=8.8 Hz, 1H, H^N (Thr)), 12.31 (br, 1H, H^COO (Asp)).

Minor conformer 0.86 (d, ³J=6.9 Hz, 3H, H^γ (Val)), 0.92 (d, ³J=6.9 Hz, 3H, H^γ (Val)), 0.96 (d, ³J=6.3 Hz, 3H, H^γ(Thr)), 1.74 (m, 1H, H^β(Pro)), 1.80-1.90 (m, 2H, H^β (Gln)), 1.90-2.02 (m, 3H, 2H^γ(Pro), H^β(Val)), 2.06-2.31 (m, 3H, 2H^γ (Gln), H^β

(Pro)), 2.91 (dd, ²J=16.3 Hz, ³J=5.0 Hz, 1H, H^β(Asp)), 2.64 (dd, ²J=16.0 Hz, ³J=7.9 Hz, 1H, H^β(Asp)), 3.30-3.67 (br, 3H, 2H^δ(Pro), H^β(Ser)), 3.69 (m, 1H, H^α(Val)), 3.80 (ddd, ³J=6.4 Hz, ³J=6.4 Hz, ³J=3.8 Hz, 1H, H^α(Gln)), 3.90 (m, 1H, H^β(Ser)), 4.15 (dd, ³J=9.4 Hz, ³J=1.9 Hz, 1H, H^α(Pro)), 4.18-4.24 (m, 2H, H^α(Thr), H^β(Thr)), 4.30 (m, 1H, H^α(Asp)), 4.72 (ddd, ³J=6.8 Hz, ³J=3.3 Hz, ³J=3.3 Hz, 1H, H^α(Ser)), 4.90-5.10 (br, 2H, H^O (Ser), H^O (Thr)), 6.88 (s, 1H, H^N (Gln)), 7.24 (m, 1H, H^N (Asp)), 7.45 (d, ³J=6.9 Hz, 1H, H^N (Thr)), 7.78 (d, ³J=6.9 Hz, 1H, H^N (Ser)), 8.22 (d,

3J=5.0 Hz, 1H, H^N (Val)), 8.45 (br, 1H, H^N (Gln)), 8.83 (s, 1H, H^N (Gln)), 12.31 (br, 1H, H^COO (Asp)).

c-(-Thr-Gln-Val-Asp-Ser-ββββ-hPro-) (53)

C27H43N7O11 (641.69 g/mol).

Cyclization on resin:

The linear peptide H-Ser(t-Bu)-β-hPro-Thr(t-Bu)-Gln(Trt)-Val-Asp(Wang)-ODmb

(110) is synthesized and cyclized following protocol 6 (3.0 equiv HATU and 6.0 equiv DIPEA as coupling reagents) on Wang resin (330 mg, 0.2 mmol). After complete deprotection and cleavage from the resin with the cleavage cocktail TFA/DCM/TIS = 48 : 48: 4 during 2 h (protocol 10), the cyclic peptide (53) is purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (53)): 10 mg, 16 µmol, 8 %.

HPLC (anal. method 2): tR=13.0 min, 100 area %.

Cyclization in solution:

The linear peptide H-Gln(Trt)-Val-Asp(Ot-Bu)-Ser(t-Bu)-β-hPro-Thr(t-Bu)-OH (111)

is synthesized on 2-ClTrt resin (250 mg, 0.20 mmol) previously loaded with Fmoc-Thr(t-Bu)-OH (loading 0.80 mmol/g), and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (111)): 160 mg, 0.13 mmol, 67 %.

HPLC (anal. method 1): tR=min, 90 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1070.62 [M+H]⁺, 1092.60 [M+Na]⁺, 1108.57 [M+K]⁺ Found: 1092.61 [M+Na]⁺

The linear peptide (111) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (111) (160 mg, 0.13 mmol) in 10 ml DMF.

Second syringe: Solution of HATU (3.0 equiv, 0.4 mmol, 153 mg,) in 10 ml DMF.

Addition rate for both syringes: 0.04 ml/min.

Flask: Solution of DIPEA (6.0 equiv, 0.80 mmol, 104 mg, 137 µl) and HATU (0.1 equiv, 13 µmol, 5 mg) in 20 ml DMF.

After evaporation of the solvent the cyclic protected peptide (112) is purified by reverse phase preparative HPLC.

Yield (cyclic protected peptide (112)): 84 mg, 50 µmol, 37 %.

HPLC (anal. method 1): tR=35.8 min, 62 area % sum of epimers.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1052.61 [M+H]⁺, 1074.59 [M+Na]⁺, 1090.56 [M+K]⁺ Found: 1074.72 [M+Na]⁺

The cyclic protected peptide (112) is completely deprotected with reagent K during 2 h (protocol 13) and finally purified by preparative HPLC.

Yield (cyclic deprotected peptide (53)): 27 mg, 42 µmol, 84 %.

HPLC (anal. method 2): tR=11.4 min, 11.7 min, 100 area %, epimers ratio 1:1.

For the biological tests and the NMR analysis the epimers were separated by reverse phase preparative HPLC (preparative method 3).

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 642.31 [M+H]⁺, 664.29 [M+Na]⁺, 680.27 [M+K]⁺ Found: 642.54 [M+H]⁺, 664.68 [M+Na]⁺, 680.58 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two sets of signals (ratio 3 : 1).

Major conformer 0.87 (d, ³J=6.3 Hz, 3H, H^γ(Val)), 0.94 (d, ³J=6.3 Hz, 3H, H^γ(Val)), 1.00 (d, ³J=6.9 Hz, 3H, H^γ(Thr)), 1.61 (dddd, ²J=12.4 Hz, ³J=7.9 Hz, ³J=7.9 Hz,

3J=7.5 Hz, 1H, H^γ (β-hPro)), 1.72-1.88 (m, 4H, 2H^β(Gln), 2H^δ(β-hPro)), 1.92 (m, 1H, H^β(Val)), 2.07 (dddd, ²J=12.2 Hz, ³J=6.2 Hz, ³J=6.2 Hz, ³J=6.2 Hz, 1H, H^γ(β -hPro)), 2.11-2.18 (m, 2H, H^γ (Gln)), 2.21 (dd, ²J=14.8 Hz, ³J=1.6 Hz, 1H, H^α(β -hPro)), 2.49 (br, 1H, H^β(Asp)), 2.67-2.76 (m, 2H, H^β(Asp), H^α(β-hPro)), 3.26-3.68 (br, 5H, 2H^ε (β-hPro), 2H^β (Ser), H^α (Val)), 4.11 (m, 1H, H^β (β-hPro)), 4.19 (dd,

3J=9.4 Hz, ³J=1.3 Hz, 1H, H^α(Thr)), 4.36-4.47 (m, 2H, H^α (Asp), H^β (Thr)), 4.55 (ddd, ³J=7.2 Hz, ³J=7.2 Hz, ³J=5.0 Hz, 1H, H^α (Ser)), 4.63 (ddd, ³J=9.1 Hz,

3J=10.1Hz, ³J=2.2 Hz, 1H, H^α(Gln)), 6.82 (s, 1H, H^N (Gln)), 7.00 (d, ³J=6.9 Hz, 1H, H^N (Ser)), 7.07 (s, 1H, H^N (Gln)), 7.49 (d, ³J=10.0 Hz, 1H, H^N (Thr)), 7.97 (d J=8.8 Hz, 1H, H^N (Asp)), 8.18 (d, ³J=8.8 Hz, 1H, H^N (Gln)), 8.28 (d, ³J=3.1 Hz, 1H, H^N (Val)), 12.33 (br, 1H, H^COO (Asp)).

Minor conformer 0.77 (d, ³J=6.9 Hz, 3H, H^γ(Val)), 0.81 (d, ³J=6.9 Hz, 3H, H^γ(Val)), 1.08 (d, ³J=6.3 Hz, 3H, H^γ(Thr)), 1.72-1.98 (m, 6H, 2H^γ(β-hPro), 2H^δ(β-hPro), 2H^β (Gln)), 2.11-2.24 (m, 3H, 2H^γ(Gln), H^β(Val)), 2.47-2.57 (br, 2H, H^β(Asp)), 2.39 (dd,

2J=14.4 Hz, ³J=8.2 Hz, 1H, H^α(β-hPro)), 2.99 (dd, ²J=14.1 Hz, ³J=2.2 Hz, 1H, H^α (β-hPro)), 3.17 (ddd, ²J=11.8 Hz, ³J=8.3 Hz, ³J=8.3 Hz, 1H, H^ε(β-hPro)), 3.26-3.68

(br, 3H, H^ε(β-hPro), 2H^β(Ser)), 3.86 (dd, ³J=8.8 Hz, ³J=6.9 Hz, 1H, H^α(Val)), 3.97 (ddd, ³J=7.9 Hz, ³J=5.7 Hz, ³J=5.7 Hz, 1H, H^α(Gln)), 4.29-4.35 (m, 2H H^α(Thr), H^β (Thr)), 4.42 (m, 2H, H^α(Asp), H^β(β-hPro)), 4.76 (ddd, ³J=8.5 Hz, ³J=6.3 Hz, ³J=6.3 Hz, 1H, H^α(Ser)), 6.81 (s, 1H, H^N (Gln)), 7.27 (d, ³J=6.9 Hz, 1H, H^N (Asp)), 7.30 (s, 1H, H^N (Gln)), 7.46 (d, ³J=8.8 Hz, 1H, H^N (Val)), 7.67 (d, ³J=9.4 Hz, 1H, H^N (Thr)), 8.25 (d, ³J=5.7 Hz, 1H, H^N (Gln)), 8.43 (d, ³J=6.3 Hz, 1H, H^N (Ser)), 12.33 (br, 1H, H^COO (Asp)).

c-(-Thr-Gln-D-Val-Asp-Ser-Pro-) (54)

C26H41N7O11 (627.66 g/mol).

Cyclization on resin:

The linear peptide H-Ser(t-Bu)-Pro-Thr(t-Bu)-Gln(Trt)-D-Val-Asp(Wang)-ODmb

(113) is synthesized and cyclized following protocol 6 (HATU as coupling reagent) on Wang resin (400 mg, 0.20 mmol). The cyclic peptide (54) is completely deprotected, cleaved from the resin with the cleavage cocktail DCM/TFA/TIS = 48 : 48 : 4 (protocol 10), and purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (54)): 10 mg 14 µmol, 7 %.

HPLC (anal. method 2): tR=9.9 min, 90 area %.

Cyclization in solution:

The linear peptide H-D-Val-Asp(Ot-Bu)-Ser(t-Bu)-Pro-Thr(t-Bu)-Gln(Trt)-OH (114) is synthesized on 2-ClTrt resin (400 mg, 0.20 mmol) previously loaded with Fmoc-Gln(Trt)-OH (loading 0.50 mmol/g), cleaved with 1 % TFA in DCM (protocol 9), and purified by preparative HPLC.

Yield (linear protected peptide (114)): 160 mg, 0.13 mmol, 65 %.

HPLC (anal. method 1): tR=29.2 min, 85 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1056.60 [M+H]⁺, 1078.58 [M+Na]⁺, 1094.56 [M+K]⁺ Found: 1056.42 [M+H]⁺, 1078.31 [M+Na]⁺, 1094.45 [M+K]⁺ The linear peptide (114) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (114) (160 mg, 0.13 mmol) in 15 ml DMF.

Second syringe: Solution of HATU (1.1 equiv, 53 mg, 0.14 mmol) in 15 ml DMF.

Addition rate for both syringes: 0.01 ml/min.

Flask: Solution of DIPEA (3.0 equiv, 0.39 mmol, 50 mg, 67 µl) and HOAt (1.0 equiv, 0.13 mmol, 17 mg) in 20 ml DMF.

After evaporation of the solvent the epimers are separated by reverse phase preparative HPLC.

Yield (cyclic protected peptide (115)): 52 mg, 47 µmol, 36 %.

HPLC (anal. method 1): tR=32.8 min, 93 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 1038.59 [M+H]⁺, 1060.57 [M+Na]⁺, 1076.55 [M+K]⁺ Found: 1060.29 [M+Na]⁺, 1076.85 [M+K]⁺

The cyclic protected peptide (115) is completely deprotected with reagent K during 5 h (protocol 13) and finally purified by preparative HPLC.

Yield (cyclic deprotected peptide (54)): 27 mg, 43 µmol, 92 %.

HPLC (anal. method 2): tR= 9.9 min, 100 area %. No isomers observed.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 628.29 [M+H]⁺, 650.28 [M+Na]⁺, 666.25 [M+K]⁺ Found: 628.27 [M+H]⁺, 650.32 [M+Na]⁺, 666.21 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two sets of signals (ratio 5 : 1).

0.85 (d, ³J=6.3 Hz, 3H, H^γ (D-Val)), 0.97 (d, ³J=6.9 Hz, 3H, H^γ (D-Val)), 1.01 (d,

3J=6.9 Hz, 3H, H^γ(Thr)), 1.68-1.81 (m, 2H, H^β(Gln), H^β(Pro)), 1.83-2.02 (m, 6H, 2H^γ(Gln), H^β(Gln), 2H^γ(Pro), H^β(D-Val)), 2.24 (m, 1H, H^β(Pro)), 2.45 (dd, ²J=16.8 Hz, ³J=9.0 Hz, 1H, H^β(Asp)), 2.83 (dd, ²J=16.6 Hz, ³J=4.7 Hz, 1H, H^β(Asp)), 3.56 (dd, ³J=10.1 Hz, ³J=5.0 Hz, 1H, H^α (D-Val)), 3.68 (ddd, ²J=9.7 Hz, ³J=9.7 Hz,

3J=6.3 Hz, 1H, H^δ(Pro)), 3.76 (ddd, ²J=9.9 Hz, ³J=8.0 Hz, ³J=2.4 Hz, 1H, H^δ(Pro)), 3.82 (dd, ²J=11.9 Hz, ³J=2.5 Hz, 1H, H^β (Ser)), 3.95 (m, 1H, H^β(Thr)), 4.00 (dd,

2J=11.6 Hz, ³J=6.0 Hz, 1H, H^β(Ser)), 4.04 (dd, ³J=9.4 Hz, ³J=3.8 Hz, 1H, H^α(Thr)), 4.07 (dd, ³J=9.1 Hz, ³J=7.9 Hz, 1H, H^α (Pro)), 4.32 (ddd, ³J=7.7 Hz, ³J=5.2 Hz,

3J=4.2 Hz, 1H, H^α (Gln)), 4.67 (ddd, ³J=9.0 Hz, ³J= 9.0 Hz, ³J= 4.6 Hz, 1H, H^α (Asp)), 4.74 (ddd, ³J=8.6 Hz, ³J=5.8 Hz, ³J=2.7 Hz, 1H, H^α(Ser)), 4.91 (br, 1H, H^O (Thr)), 5.29 (br, 1H, H^O (Ser)), 6.63 (br, 1H, H^N (Gln)), 6.70 (d, ³J=5.0 Hz, 1H, H^N (Gln)), 6.99 (d, ³J=9.4 Hz, H^N (Thr)), 7.16 (br, 1H, H^N (Gln)), 7.99 (d, ³J=8.2 Hz, 1H, H^N (Ser)), 8.68 (d, ³J=8.8 Hz, H^N (Asp)), 8.87 (d, ³J =5.0 Hz, H^N (Val)), 12.84 (br, 1H, H^COO (Asp)).

c-(-Gln-Ile-Asp-Ser-Pro-) (55)

C23H36N6O9 (540.58 g/mol).

Cyclization on resin:

The linear peptide H-Ser(t-Bu)-Pro-Gln(Trt)-Ile-Asp(Wang)-ODmb (116) is synthesized and cyclized following protocol 6, with different coupling reagents (Table 37), on Wang resin. After complete deprotection and cleavage from the resin, with different cleavage cocktails (Table 37), the cyclic peptide (55) is purified by reverse phase preparative HPLC.

Table 37: On resin cyclization of H-Ser(t-Bu)-Pro-Gln(Trt)-Ile-Asp(Wang)-ODmb (116)

Resin n/

mmol Coupling reagent t/

min

Cleavage Reagent

t/

h Yield HPLC/

% Wang

135 mg 0.10

1.1 equiv HATU 3.0 equiv DIPEA

2.0 equiv HOAt

45 Reagent B 5

6 mg 8 µmol

8 %

70^a

Wang

130 mg 0.10 3.0 equiv HATU 6.0 equiv DIPEA 40

TFA 47 DCM 47

TIS 6

2

3 mg 6 µmol

6 %

100^a

a The same HPLC analytical method and the retention time as for the cyclization in solution.

Cyclization in solution:

The linear peptide H-Ile-Asp(Ot-Bu)-Ser(t-Bu)-Pro-Gln(Trt)-OH (117) is synthesized on 2-ClTrt resin (300 mg, 0.20 mmol) previously loaded with Fmoc-Gln(Trt)-OH (loading 0.66 mmol/g), and cleaved with 1 % TFA in DCM (protocol 9).

Yield (linear protected peptide (117)): 150 mg, 89 µmol, 45 %.

HPLC (anal. method 1): tR= 26.9 min, 54 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 913.51 [M+H]⁺, 935.49 [M+Na]⁺, 951.46 [M+K]⁺ Found: 913.33 [M+H]⁺, 935.28 [M+Na]⁺, 951.36 [M+K]⁺ The linear peptide (117) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (117) (150 mg, 89 µmol) in 10 ml DMF.

Second syringe: Solution of HATU (6.0 equiv, 0.53 mmol, 200 mg) in 10 ml DMF.

Addition rate for both syringes: 0.01 ml/min.

Flask: Solution of DIPEA (13.0 equiv, 1.17 mmol, 151 mg, 200 µl) in 20 ml DMF.

After evaporation of the solvent the epimers are separated by reverse phase preparative HPLC.

Yield (cyclic protected peptide (118)): 29 mg, 26 µmol, 29 %.

HPLC (anal. method 1): tR= 31.8 min, 81 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 895.50 [M+H]⁺, 917.48 [M+Na]⁺, 933.45 [M+K]⁺ Found: 917.28 [M+Na]⁺, 933.45 [M+K]⁺

The protected cyclic peptide (118) is completely deprotected with reagent K during 5 h (protocol 13) and finally purified by preparative HPLC.

Yield (cyclic deprotected peptide (55)): 15 mg, 24 µmol, 92 %.

HPLC (anal. method 2): tR= 11.0 min, 85 area %. No isomers observed.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 541.26 [M+H]⁺, 563.24 [M+Na]⁺, 579.22 [M+K]⁺ Found: 541.49 [M+H]⁺, 563.69 [M+Na]⁺, 579.71 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two sets of signals (ratio 3 : 1).

Major conformer: 0.81 (d, ³J=6.3 Hz, 3H, H^γ(Ile)), 0.82 (dd, ³J=7.5 Hz, ³J=7.5 Hz, 3H, H^δ(Ile)), 0.98 (ddq, ²J=13.8 Hz, ³J=7.2 Hz, ³J=1.3 Hz, 1H, H^γ(Ile)), 1.38 (m, 1H, H^γ(Ile)), 1.65-1.79 (m, 2H, H^β(Gln), H^γ(Pro)), 1.79-1.92 (m, 3H, H^β(Gln), H^β (Ile), H^γ (Pro)), 1.92-2.09 (m, 3H, 2H^γ (Gln), H^β (Pro)), 2.21 (dddd, ²J=10.4 Hz,

3J=9.8 Hz, ³J=7.5 Hz, ³J=2.8 Hz, 1H, H^β(Pro)), 2.33 (dd, ²J=16.9 Hz, ³J=11.3 Hz, 1H, H^β(Asp)), 2.71 (dd, ²J=16.6, ³J=2.2 Hz, 1H, H^β(Asp)), 3.36 (dd, ²J=10.1 Hz,

3J=5.0 Hz, 1H, H^β(Ser)), 3.39-3.56 (br, 2H, H^δ(Pro), H^β(Ser)), 3.60 (ddd, ²J=11.5 Hz, ³J=7.7 Hz, ³J=3.6 Hz, 1H, H^δ(Pro)), 4.11 (ddd, ³J=10.8 Hz, ³J=10.8 Hz, ³J=2.6 Hz, 1H, H^α(Asp)), 4.30 (ddd, ³J=9.9 Hz, ³J=9.9 Hz, ³J=5.5 Hz, 1H, H^α(Gln)), 4.40 (ddd, ³J=8.6 Hz, ³J=8.6 Hz, ³J=5.2 Hz, 1H, H^α(Ser)), 4.47 (dd, ³J=9.1 Hz, ³J=9.1 Hz, 1H, H^α(Ile)), 4.51 (dd, ³J=8.8 Hz, ³J=2.5 Hz, 1H, H^α(Pro)), 6.77 (s, 1H, H^N (Gln)), 6.94 (d, ³J=8.2 Hz, 1H, H^N (Ser)), 7.28 (s, 1H, H^N (Gln)), 7.54 (d, ³J=10.0 Hz, 1H, H^N (Gln)), 7.84 (d, ³J=10.7 Hz, 1H, H^N (Asp)), 8.35 (d, ³J=9.4 Hz, 1H, H^N (Ile)), 12.51 (br, 1H, H^COO (Asp)).

Minor conformer 0.78 (d, ³J=6.3 Hz, 3H, H^γ(Ile)), 0.79-0.83 (m, 3H, H^δ(Ile)), 1.04 (m, 1H, H^γ(Ile)), 1.42 (m, 1H, H^γ(Ile)), 1.70 (m, 1H, H^γ(Pro)), 1.79-1.92 (m, 4H, 2H^β (Gln), H^β (Ile), H^γ (Pro)), 1.92-2.09 (m, 4H, 2H^γ (Gln), 2H^β (Pro)), 2.74 (dd,

2J=16.0 Hz, ³J=6.0 Hz, 1H, H^β (Asp)), 2.80 (dd, ²J=16.0 Hz, ³J=7.5 Hz, 1H, H^β (Asp)), 3.39-3.56 (br, 3H, 2H^δ(Pro), H^β(Ser)), 3.69 (dd, ²J=10.1 Hz, ³J=4.4 Hz, 1H, H^β (Ser)), 3.75 (dd, ³J=8.7 Hz, ³J=8.5 Hz, 1H, H^α (Ile)), 4.10 (m, 1H, H^α(Ser)), 4.19-4.27 (m, 2H, H^α (Asp), H^α (Gln)), 4.59 (dd, ³J=6.6 Hz, ³J=2.2 Hz, 1H, H^α (Pro)), 6.82 (s, 1H, H^N (Gln)), 7.33 (s, 1H, H^N (Gln)), 7.66 (d, ³J=3.8 Hz, 1H H^N

(Ser)), 7.73 (d, ³J=8.8 Hz, 1H H^N (Gln)), 7.94 (d, ³J=8.2 Hz, 1H H^N (Ile)), 8.02 (d,

3J=8.8 Hz, 1H H^N (Asp)), 12.51 (br, 1H, H^COO (Asp)).

c-(-Gln-Ile-Asp-Ser-ββββ-hPro-) (56)

C24H38N6O9 (554.61 g/mol).

Cyclization on resin:

The linear peptide H-Ser(t-Bu)-β-hPro-Gln(Trt)-Ile-Asp(Wang)-ODmb (119) is synthesized and cyclized following protocol 6 (HATU as coupling reagent), on Wang resin (125 mg, 50 µmol), After complete deprotection and cleavage from the resin with cocktail mixture TFA/DCM/TIS = 48 : 48 : 4 (protocol 10), the cyclic peptide (56) is purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (56)): 4 mg, 7.2 µmol, 14 %.

HPLC (anal. method 2): tR= 13.1 min, 100 %.

Cyclization in solution:

The linear peptide H-β-hPro-Gln(Trt)-Ile-Asp(Ot-Bu)-Ser(t-Bu)-OH (120) is synthesized on 2-ClTrt resin (200 mg, 0.20 mmol) previously loaded with Fmoc- -Ser(t-Bu)-OH (loading 1.00 mmol/g), and cleaved with 1 % TFA in DCM (protocol 9).

Yield (protected linear peptide (120)): 74 mg, 72 µmol, 36 %.

HPLC (anal. method 1): tR= 28.1 min, 90 %.

MALDI-ToF MSmonoisotopic, m/z

Calcd.: 927.52 [M+H]⁺, 949.51 [M+Na]⁺, 965.50 [M+K]⁺ Found: 927.49 [M+H]⁺

The linear peptide (120) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (120) (74 mg, 72 µmol) in 10 ml DMF.

Second syringe: Solution of HATU (3.0 equiv, 0.22 mmol, 82 mg) in 10 ml DMF.

Addition rate for both syringes: 0.05 ml/min.

Flask: Solution of DIPEA (6.0 equiv, 0.43 mmol, 56 mg, 74 µl) and HATU (0.1 equiv, 7 µmol 3 mg) in 10 ml DMF.

After evaporation of the solvent the the epimers are separated by reverse phase preparative HPLC.

Yield (cyclic protected peptide (121)): 40 mg, 26 µmol, 36 %.

HPLC (anal. method 1): tR= 32.7 min, 60 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 909.51 [M+H]⁺, 931.49 [M+Na]⁺, 947.47 [M+K]⁺ Found: 931.54 [M+Na]⁺, 947.62 [M+K]⁺

The cyclic protected peptide (121) is completely deprotected with reagent K during 2 h (protocol 13) and finally purified by reverse phase preparative HPLC.

Yield (cyclic deprotected peptide (56)): 13 mg, 23 µmol, 89 %.

HPLC (anal. method 2): tR= 13.1 min, 100 area %. No isomers observed.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 555.28 [M+H]⁺, 577.26 [M+Na]⁺, 593.23 [M+K]⁺ Found: 555.26 [M+H]⁺, 577.08 [M+Na]⁺, 593.00 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two sets of signals (ratio 2 : 1).

Major conformation 0.73-0.88 (m, 6H, 3H^γ (Ile), 3H^δ(Ile)), 0.98 (ddq, ²J=14.4 Hz,

3J=7.2 Hz, ³J=7.2 Hz, 1H, H^γ(Ile)), 1.35 (m, 1H; H^γ(Ile)), 1.62 (m, 1H, H^γ(Gln)), 1.76-1.91 (m, 5H, H^β(Ile), 2H^γ (β-hPro), 2H^δ (β-hPro)), 1.90-2.10 (m, 2H, H^β(Gln), H^γ(Gln)), 2.17 (m, 1H, H^β(Gln)), 2.34 (dd, ²J=17.3 Hz, ³J=6.6 Hz, 1H, H^α (β-hPro)), 2.55 (dd, ²J=16.6 Hz, ³J=8.5 Hz, 1H, H^β (Asp)), 2.60 (dd, ²J=17.6 Hz, ³J=2.5 Hz, 1H, H^α (β-hPro)), 2.73 (dd, ²J=16.3 Hz, ³J=5.7 Hz, 1H, H^β (Asp)), 3.27-3.55 (br, 2H, H^ε (β-hPro)), 3.64 (dd, ²J=12.6 Hz, ³J=4.4 Hz, 1H, H^{β (Ser)}), 3.82 (dd, ²J=12.6 Hz,

3J=3.1 Hz, 1H, H^{β (Ser)}), 3.87 (dd, ³J=9.7 Hz, ³J=9.7 Hz, 1H, H^α(Ile)), 4.06 (m, 1H, H^α(Gln)), 4.39 (m, 1H, H^α(Ser)), 4.50 (ddd, ³J=8.5 Hz, ³J=8.5 Hz, 6.3 Hz, 1H, H^α (Asp)), 4.60 (m, 1H, H^β (β-hPro)), 6.72 (s, 1H, H^N (Gln)), 7.22 (s, 1H, H^N (Gln)), 7.33 (br, 1H, H^N (Gln)), 7.66 (d, ³J=9.4 Hz, 1H, H^N (Ile)), 7.70 (d, ³J=8.8 Hz, 1H, H^N (Asp)), 7.97 (d, ³J=6.9 Hz, 1H, H^N (Ser)), 12.3 (br, 1H, H^COO (Asp)).

Minor conformation 0.73-0.88 (m, 6H, 3H^γ (Ile), 3H^δ(Ile)), 0.98 (ddq, ²J=14.4 Hz,

3J=7.2 Hz, ³J=7.2 Hz, 1H, H^γ(Ile)), 1.35 (m, 1H, H^γ(Ile)), 1.48 (m, 1H, H^δ(β-hPro)), 1.59-1.76 (m, 2H, H^β(Gln), H^γ(β-hPro)), 1.76-1.91 (m, 2H, H^β(Gln), H^δ(β-hPro)), 1.90-2.10 (m, 5H, 2H^γ (Gln), H^β (Ile), H^α (β-hPro), H^γ (β-hPro)), 2.17 (m, 1H, H^α (β-hPro)), 2.92 (m, 1H, H^ε (β-hPro)), 2.89 (dd, ²J=16.3 Hz, ³J=8.8 Hz, 1H, H^β (Asp)), 2.95 (dd, ²J=16.6 Hz, ³J=5.3 Hz, 1H, H^β (Asp)), 3.27-3.55 (br, 2H, H^ε (β -hPro), H^β (Ser)), 3.58 (dd, ²J=17.0 Hz, ³J=6.9 Hz, 1H, H^β (Ser)), 3.93-4.01 (m, 2H, H^α(Asp), H^α(Ile)), 4.12 (ddd, ³J=9.1 Hz, ³J=9.1 Hz, ³J=5.0 Hz, 1H, H^α(Gln)), 4.31 (m, 1H, H^β(β-hPro)), 4.45 (m, 1H, H^α(Ser)), 6.81 (s, 1H, H^N (Gln)), 7.33 (br, 1H, H^N (Gln)), 7.66 (d ³J=8.2 Hz, 1H, H^N (Ile)), 7.73 (d, ³J=9.3 Hz, 1H, H^N (Gln)), 7.98 (d,

3J=5.7 Hz, 1H, H^N (Ser)), 9.21 (d, ³J=6.9 Hz, 1H, H^N (Asp)), 12.3 (br, 1H, H^COO (Asp)).

c-(-Gln-Leu-Asp-Ser-Pro-) (57)

C23H36N6O9 (540.58 g/mol).

Cyclization in solution:

The linear peptide H-Asp(Ot-Bu)-Ser(t-Bu)-Pro-Gln(Trt)-Leu-OH (122) is synthesized on 2-ClTrt resin (278 mg, 0.20 mmol) previously loaded with Fmoc- -Leu-OH (loading 0.72 mmol/g), cleaved with 1 % TFA in DCM (protocol 9) and purified by preparative HPLC.

Yield (linear protected peptide (122)): 130 mg, 0.14 mmol, 71 %.

HPLC (anal. method 1): tR= 27.4 min, 100 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 913.51 [M+H]⁺, 935.49 [M+Na]⁺, 951.46 [M+K]⁺ Found: 935.93 [M+Na]⁺, 951.52 [M+K]⁺

The linear peptide (122) is cyclized following protocol 7, method B:

First syringe: Solution of linear peptide (122) (130 mg, 0.14 mmol) in 20 ml DMF.

Second syringe: Solution of HATU (1.1 equiv, 0.16 mmol, 59 mg) in 20 ml DMF.

Addition rate for both syringes: 0.01 ml/min.

Flask: Solution of DIPEA (2.0 equiv, 0.28 mmol, 37 mg, 49 µl) and HOAt (1.0 equiv, 0.14 mmol 19 mg) in 20 ml DMF.

After evaporation of DMF the cyclic protected peptide (123) is purified by reverse phase preparative HPLC.

Yield (cyclic protected peptide (123)): 120 mg, 0.10 mmol, 71 %.

HPLC (anal. method 1): tR= 31.4 min, 75 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 895.50 [M+H]⁺, 917.48 [M+Na]⁺, 933.45 [M+K]⁺ Found: 895.76 [M+H]⁺, 917.73 [M+Na]⁺, 933.70 [M+K]⁺

The cyclic protected peptide (123) is completely deprotected with reagent K during 5 h (protocol 13) and finally purified by preparative HPLC.

Yield (cyclic deprotected peptide (57)): 32 mg, 44 µmol, 44 %.

HPLC (anal. method 2): tR= 11.9 min, 75 area %.

MALDI-ToF MSmonoisotopic, m/z:

Calcd.: 541.26 [M+H]⁺, 563.24 [M+Na]⁺, 579.22 [M+K]⁺ Found: 541.27 [M+H]⁺, 563.43 [M+Na]⁺, 579.32 [M+K]⁺

1H NMR (500 MHz, DMSO-d6), δ (ppm): Two sets of signals (ratio 9 : 1).

0.84 (d, ³J=6.9 Hz, 3H, H^δ (Leu)), 0.86 (d, ³J=6.3 Hz, 3H, H^δ (Leu)), 1.15 (ddd,

2J=13.7 Hz, ³J=6.8 Hz, ³J=6.4 Hz, 1H, H^β(Leu)), 1.47 (m, 1H, H^γ(Leu)), 1.62-1.76 (m, 4H, 2H^β (Gln), H^β (Leu), H^γ (Pro)), 1.84 (m, 1H, H^γ (Pro)), 1.88-2.00 (m, 3H, 2H^γ (Gln), H^β(Pro)), 2.19 (dddd, ²J=16.3 Hz, ³J=9.3 Hz, ³J=6.8 Hz, ³J=6.8 Hz, 1H,

Im Dokument [beta]-Amino acids as secondary structure inducers in peptides (Seite 139-175)